As others have described, the fibers of the orbital region were
more complex. In most sections along the muscle, most orbital fibers
reacted with the antibody to the embryonic MyHC (
Fig. 4B ). In one discrete area, however, most likely the region of the
neuromuscular junction (see later discussion), almost all fibers failed
to react with this antibody
(Fig. 4A) . Conversely, at this same area,
most orbital fibers reacted with the antibody to the EOM-specific MyHC
(Fig. 4C) . Several sections distally, however, staining with this
antibody began to fade from most fibers
(Fig. 4D) . In general, fibers
in the global region did not react with the antibodies to embryonic or
extraocular MyHCs and neither did fibers in the levator palpebrae.
Occasional scattered fibers in the global region showed some
inconsistent reaction with one or the other of these antibodies.
Because the great majority of the orbital fibers reacted with both
these antibodies, we equate them with the orbital singly innervated
fibers (oSIFs) which reportedly comprise 80% of orbital
fibers.
3 A smaller percentage of fibers did not react with
anti-extraocular MyHC antibody, but instead reacted with the
anti-slow-twitch MyHC antibody
(Fig. 1C) and showed no longitudinal
variation in staining with this antibody. Away from the neuromuscular
junction, these reacted with the anti-embryonic MyHC antibody. These
are presumably the oMIFs. A small number of fibers, mostly close to the
orbital–global border, stained with antibody to the IIa MyHC
(Fig. 1B) . These fibers did not react with antibody to the slow-twitch MyHC,
but reacted with embryonic MyHC in the same proximal–distal manner
that other orbital fibers reacted and with antibody to the EOM-specific
isoform near the motor endplate
(Fig. 4D) .
To confirm and to delineate more precisely the longitudinal variation
of MyHC isoforms along orbital fibers, we combined fluorescent antibody
staining of whole EOMs with confocal microscopy.
Figure 5A shows the orbital region of a rat EOM stained with the antibody to
embryonic MyHC. This antibody stained a majority of fibers in the
orbital region, as seen in the cross sections; however, there was a
sharp exclusion of staining at the putative neuromuscular junction
region, although staining was present throughout the remainder of the
fiber.
Staining a whole muscle with antibody to the extraocular MyHC gave an
approximate complementary pattern of localization
(Fig. 5B) . Reactivity
occurred most strongly at the putative neuromuscular junction and in a
wide swath to either side, then rapidly faded toward the end of the
muscle layer. The domain occupied by EOM-MyHC was not so sharply
delineated as was the embryonic MyHC. A possible correlation of
previously described orbital fiber types and their MyHC isoforms is
shown in
Table 1 .
The area devoid of staining with the anti-embryonic MyHC antibody
(Fig. 5A) was related to the neuromuscular junction. Under the light
microscope, we could visualize the motoneuron entering this specific
region. To confirm this, we double stained EOMs with FITC-labeled
anti-embryonic MyHC antibody (
Fig. 6A ) and with Texas red-labeled Ta51, a monoclonal antibody to
neurofilaments
(Fig. 6B) .
Figure 6C is a superimposition of
Figures 6A and 6B . This shows the putative neuromuscular junction area with
embryonic MyHC positive fibers approaching from both sides. The great
majority of neurofilaments were located in the region devoid of
staining with the anti-embryonic MyHC antibody, strongly suggesting
that this region is the location of the neuromuscular junction.
Staining with Texas red–labeled α-bungarotoxin confirmed this
finding (not shown).
The orbital fibers which were positive for embryonic MyHC continue into
the neuromuscular junction region, and the cessation of anti-embryonic
MyHC staining did not mark the end of the fibers.
Figures 5C 5D and 5E show an enlargement of fibers at the edge of the neuromuscular
junction region, approximately the area within the box in
Figure 5A .
Muscles were double stained with FITC-labeled anti-embryonic MyHC
antibody
(Fig. 5C) and with Texas red–labeled phalloidin
(Fig. 5D) .
Figure 5E is a superimposition of both stains. Staining with
anti-embryonic MyHC faded rapidly as the fibers entered the region of
the neuromuscular junction. The fibers that reacted with the
anti-embryonic MyHC antibody, however, continued into the neuromuscular
junction region, which was devoid of staining with the anti-embryonic
antibody, but remained positive with the phalloidin (long arrows).
Because others have shown that the orbital slow fibers may contain some
slow tonic MyHC, we tested a number of polyclonal and monoclonal
antibodies against the slow tonic MyHC. Unfortunately, all the slow
tonic antibodies reacted with the same fibers stained by NOQ7-5–4D,
the antibody to the slow-twitch MyHC. Therefore, we cannot
differentiate between slow-twitch and slow tonic fibers. Moreover,
staining sections with BF-34, an antibody against the neonatal MyHC did
not give clean results. All fibers, except those reacting with the
antibody to slow-twitch MyHC, reacted with BF-34. Unfortunately, other
skeletal muscles showed the same pattern of staining, even though PCR
did not identify the neonatal MyHC mRNA in those muscles. Therefore, it
is also not possible to localize the neonatal isoform, although others
have suggested a distribution similar to the one we have described for
the embryonic MyHC.
8 19
Because PCR showed the presence of α-cardiac MyHC mRNA (Rubinstein,
unpublished results), we used F8812F8 to stain sections and discovered
a small population of cells, possibly oMIFs, which additionally reacted
with that antibody
(Fig. 4E) . No global region fibers in the rat
reacted with the anti-α-cardiac MyHC antibody.