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Lanthanum hydroxide labelling of gap junctions in the odontoblast layer.

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Lanthanum Hydroxide Labelling of Gap
Junctions in the Odontoblast Layer
D e p a r t m e n t of Endodontics a n d Ultrastructure Laboratory, Dental Science
Building, The University of Iowa, Iowa C i t y , Iowa 52242
Earlier work has suggested that terminal nerve fibers in the
periphery of the dental pulp are linked to one another and to odontoblasts by
gap junctions although positive identification of the junctions was lacking. In
this study, the lanthanum hydroxide tracer technique has been used to demonstrate that the junctions in this area do have the characteristic features of gap
junctions, a wide dark intermediate line in transverse section, stippling in oblique
section and a globular arrangement of sub-units in tangential section. Other
studies have shown these junctions to be of low electrical resistance although
the functional importance of this characteristic in this situation has not been
It has recently been suggested that certain intercellular junctions in the odontoblast layer of the dental pulp may be the
site of electrical coupling between odontoblasts and nerve fibers and also between
one nerve fiber and another (Matthews and
Holland, '75 j . The junctions in previous
studies were tentatively identified as gap
junctions on the basis that the junctions
were more than twice the thickness of adjacent plasma membranes (Holland, '75).
The observations reported in this paper
aimed to establish the classification of
these junctions, an essential step since the
properties of gap junctions differ considerably from those of occludens (tight) junctions, yet their appearance in routine preparations does not (McNutt and Weinstein,
' 7 3 ) . Junction thickness may not be an
adequate criterion to distinguish these
types as variations may be due to poorly
controlled conditions of tissue fixation
(Brightman and Palay, '63).
of freshly prepared lanthanum hydroxide
was added to the osmium tetroxide and
uranyl acetate solutions and to all subsequent reagents up to the final stages of
the dehydration (Revel and Karnovsky,
'67). The blocks were embedded in araldite
and after removal of the overlying hard
tissues, thin sections of the odontoblast
layer were cut and mounted on 1 m m slot
grids with formvar support films. Some
sections were examined in the electron
microscope without further staining; other
sections were stained with uranyl acetate
and lead citrate.
Parts of the blocks treated with lanthanum hydroxide contained this electron
opaque material in the extracellular space
(fig. 1) although it was not present
throughout the whole block. This is consistent with the experience of. Revel and
Karnovsky ('67 j who attributed this to incomplete penetration and subsequent leachMATERIALS AND METHODS
ing. In areas where the extracellular spaces
Young adult cats were fixed by perfusion were clearly marked, the junctions previof 2.5% glutaraldehyde in 0.06 M phos- ously suggested to be gap junctions were
phate buffer at room temperature. The visible (fig. 1-arrow). They appeared as
canine teeth were removed and slit trans- pentilaminar structures when cut in cross
versely into 1 mm thick blocks. These were section (fig. 2) with a very broad dark
post-fixed in 2% osmium tetroxide at room intermediate line of similar dimensions to
temperature for two hours and subse- that seen in gap junctions and both darker
quently stained en bloc for one hour in
Received Jan. 22, '76. Accepted Mar. 16, '76.
2 % aqueous uranyl acetate. One percent
ANAT. REC.. 1 8 6 . 121-126.
and thicker than that seen in occludens
junctions (Revel and Karnovsky, '67 ).
Whcn cut tangentially (figs. 3, 4 ) electronopaque material delineates globular subunits within the junctions similar to those
described by Revel and Karnovsky ('67)
although the hexagonal shape of the units
was not clear. A stippled pattern also found
in gap junctions (Revel and Karnovsky,
'67) was present where the junction was
sectioned obliquely (fig. 4-arrow).
By these three points, the lanthanum hydroxide tracer technique establishes these
structures as gap junctions. Several problems still exist in relating this finding to
function. A considerable body of evidence
supports the view that the gap junction is
of low electrical resistance (Bennett, '66)
and responsible for electrical coupling in
cardiac (Barr et al., '65) and smooth
(Barr et al., '68) muscle. They also occur,
however, between nonexcitablc cells such
as hepatocytes (Penn, '66). While Matthews ('75) has demonstrated electrical
coupling between pulpal nerve fibers, no
evidence is available as yet to suggest
that the odontoblast is either excitable or
coupled electrically to nerve fibers nor are
the structures forming the junctions positively identified as nerve fibers. Naked
axon terminals in such a situation cannot
be recognized morphologically, although
recent work has labelled axons in this area
autoradiographically and some of the labelled structures make specialized junctions on odontoblasts (Byers and Kish, '76).
In this study gap junctions were not seen
between adjacent odontoblast cell bodies
but further studies are in progress utilizing serial sectioning to establish the nature
of the cells and the processes involved in
these junctions.
With all these bases for doubt it is too
soon, perhaps, to form a durable hypothesis
relating these findings to the initiation of
sensory nerve activity in the tooth but sufficient evidence is now available to stimulate further studies on the possible role of
gap junctions between nerve fibers and
cells of possible receptor function as well
as between one nerve fiber and another.
I would like to thank Dr. D. Holmes for
his technical assistance.
This work was supported by USPHS
Barr, L.. W. Berger a n d M. M. Dewey 1968
Electrical transmission at the nexus between
smooth muscle cells. J. Gen. Physiol., 51: 347.
Barr, L., M. M. Dewey a n d W Berger 1965
Propagation of action potentials and the structure of t h e nexus i n cardiac muscle. J. Gen.
Physiol., 48: 797.
Bennett, M. V. L. 1966 Physiology o f electrotonic junctions. Ann. N. Y. Acad. Sci., 137:
Brightman, M. W., and S. L. Palay 1963 T h e
fine structure o f ependyma in the brain of the
rat. J. Cell B i d , 19: 415.
Byers, M. R., and S. J . Kish 1976 Delineation
of somatic nerve endings i n r a t teeth by autoradiography of axon-transported protein. J. dent.
Res., in press.
Holland, G. R . 1975 Membrane junctions o n
cat odontoblasts. Archs. oral Biol., 20: 551.
Matthews, B., and G. R. Holland 1975 Coupling between nerves i n teeth. Brain Res., 98:
McNutt, N. A,, and R. S. Weinstein 1973
Membrane ultrastructure at mammalian intercellular junctions. Progr. Biophys. mol. Biol.,
26: 45.
Penn, R. D. 1966 Ionic communication between liver cells. J. Cell Biol., 29: 171.
Revel, R. P . , and M. J. Karnovsky 1967 Hexagonal array of sub-units in intercellular junctions of the mouse heart and liver. J. Cell Biol.,
33: c 7 .
A transverse section through the odontoblast layer. Stained en bloc
with uranyl acetate and treated with lanthanum hydroxide. The arrow
labels a possble gap junction between a narrow cell process and an
odontoblast cell body. x 13,000.
One of
An enlargement of the junction arrowed i n figure 1 showing a n area,
to the left, where the junction h a s been cut tangentially and the
globular pattern of sub-units traced by the lanthanum hydroxide are
revealed. x 127,000.
Another junction cut both tangentally (left) and obliquely (arrow).
~ 2 5 5 , 0 0 0 .(All bar lines i n microns)
the specialized junctions i n the odontoblast layer cut transand labeled with lanthanum hydroxide. This section h a s been
with uranyl acetate and lead citrate to delineate the inner
of the apposed cell membranes. x 400,000.
G . R. Holland
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odontoblasts, gap, labelling, junction, layer, hydroxide, lanthanum
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