 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 2001, 21(6):1983-2000
Cell-Specific Expression of Connexins and Evidence of Restricted
Gap Junctional Coupling between Glial Cells and between Neurons
John E.
Rash1, 2,
Thomas
Yasumura1,
F. Edward
Dudek1, 2, and
James I.
Nagy3
1 Department of Anatomy and Neurobiology and
2 Program in Molecular, Cellular, and Integrative
Neurosciences, Colorado State University, Fort Collins, Colorado 80523, and 3 Department of Physiology, Faculty of Medicine,
University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7
 |
ABSTRACT |
The transmembrane connexin proteins of gap junctions link
extracellularly to form channels for cell-to-cell exchange of ions and
small molecules. Two primary hypotheses of gap junction coupling in the
CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in
both neurons and glia, and (2) intercellular junctional
coupling is restricted to specific coupling partners, with different
connexins expressed in each cell type. There is consensus that gap
junctions link neurons to neurons and astrocytes to oligodendrocytes,
ependymocytes, and other astrocytes. However, unresolved are the
existence and degree to which gap junctions occur between
oligodendrocytes, between oligodendrocytes and neurons, and between
astrocytes and neurons. Using light microscopic immunocytochemistry and
freeze-fracture replica immunogold labeling of adult rat CNS, we
investigated whether four of the best-characterized CNS connexins are
each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and
whether astrocytes share gap junctions with neurons. Connexin32 (Cx32)
was found only in gap junctions of oligodendrocyte plasma membranes,
Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only
in neurons. Oligodendrocytes shared intercellular gap junctions only
with astrocytes, with each oligodendrocyte isolated from other
oligodendrocytes except via astrocyte intermediaries. Finally, neurons
shared gap junctions only with other neurons and not with glial cells.
Thus, the different cell types of the CNS express different connexins,
which define separate pathways for neuronal versus glial gap junctional communication.
Key words:
astrocyte; connexin; connexon; gap junction; neuron; oligodendrocyte
| |
INTRODUCTION |
Astrocytes, ependymocytes, and
oligodendrocytes, the macroglial cells of the adult CNS, are richly
invested with gap junctions. Astrocytes, in particular, share gap
junctions with all three macroglia, thereby creating a functional
panglial syncytium (Mugnaini, 1986 ; Rash et al., 1997). In contrast,
gap junctions involving neurons were reported to be rare (Brightman and
Reese, 1969; Sotelo and Korn, 1978), with glial gap junctions greatly
outnumbering neuronal gap junctions and neuron-to-glial junctions not
detected (Wolff et al., 1998; Rash et al., 2000). The initial
"restricted coupling partner" hypothesis that oligodendrocytes
share intercellular gap junctions only with astrocytes and that neurons
share gap junctions only with neurons (Massa and Mugnaini, 1982;
Mugnaini, 1986; Rash et al., 1997) was supported by immunocytochemical
data showing that neurons and glia express different connexins (Li et
al., 1997; Condorelli et al., 1998; Nagy et al., 1999; Nagy and Rash,
2000; Rash et al., 2000). A quite different
"shared-connexins/mixed-coupling" hypothesis, which arose from
in situ hybridization, imaging of calcium waves, electrical
and dye coupling, and immunocytochemistry, suggests that neurons and
glia coexpress connexin32 (Cx32) and Cx43, that neuron-to-neuron and
neuron-to-glial gap junctions are abundant, and that oligodendrocytes
share gap junctions with other oligodendrocytes (Micevych and Abelson,
1991; Nedergaard, 1994; Micevych et al., 1996; Nadarajah et al., 1996;
Alvarez-Maubecin et al., 2000; for review, see Dermietzel, 1998;
Dermietzel and Spray, 1998). However, functional coupling of neurons to
glia via gap junctions has been challenged on the basis of the
demonstration of alternative signaling mechanisms between these cells
(Parpura et al., 1994; Hassinger et al., 1995; Charles et al., 1996;
Dudek et al., 1998). Moreover, the limited resolution of light
microscopy in tissue slices (~0.3-0.5 µm) precludes its use for
assigning connexins to plasma membranes of either of two apposed cells
or to intervening unresolved cell processes. Likewise, with
thin-section electron microscopy (TEM), identification of close plasma
membrane appositions as gap junctions is particularly difficult in the CNS unless strict criteria for identifying gap junctions are rigorously applied (Brightman and Reese, 1969; Sloper, 1972; Berdan et al., 1987;
Rash et al., 1998a). Although unambiguous labeling of identified gap
junctions in identified glial cells has been demonstrated (Li et al.,
1997; Nagy et al., 1999), TEM sampling methods limit quantitative
analysis of rare coupling combinations.
Freeze-fracture replica immunogold labeling [FRIL; introduced by
Fujimoto (1995)] now makes it possible to determine whether specific
connexins are present or absent in ultrastructurally defined gap
junctions in unambiguously identified neurons and glia (Rash and
Yasumura, 1999; Rash et al., 2000). Using antibodies against four well
characterized CNS connexins (Cx30, Cx32, Cx36, and Cx43), in
combination with LM immunocytochemistry and FRIL, we show that gap
junctions in astrocytes, oligodendrocytes, and neurons have
distinctive, nonshared complements of these connexins. On the basis of
cell-specific connexin distributions and correlative ultrastructural
features, we establish that oligodendrocytes share intercellular gap
junctions only with astrocytes and not detectably with other
oligodendrocytes. Furthermore, we establish that neurons share gap
junctions with neurons and not detectably with oligodendrocytes or
astrocytes. These data provide further support for the hypothesis of
restricted connexin expression and restricted gap junction-coupling pathways in neurons and in glia.
| |
MATERIALS AND METHODS |
Antibodies. Anti-connexin antibodies used in this
study (Table 1) include 12 monoclonal and
polyclonal antibodies against Cx30, Cx32, and Cx43, plus two well
characterized rabbit polyclonal antibodies against Cx36 (Rash et al.,
2000). In LM studies, oligodendrocytes were identified by the antibody
against the oligodendrocyte/myelin marker 2',3'-cyclic nucleotide
3'-phosphodiesterase [CNPase; courtesy of Dr. P. Braun, McGill
University, Montreal, Quebec, Canada; methods in Li et al. (1997)]. In
FRIL studies, identification of astrocyte plasma membranes was
confirmed with anti-aquaporin4 (AQP4) antibody, which labels AQP4
"square arrays" that are restricted to astrocyte and ependymocyte
plasma membranes (Nielsen et al., 1997; Rash et al., 1998b). In various
combinations (Table 2), these antibodies
allowed single, double, and triple labeling by FRIL, thereby
facilitating both connexin localizations and cell identifications.
Double and triple labeling were used to document the presence of one or
two connexins in one class of gap junctions, while simultaneously
documenting the presence or absence of a third connexin in the same gap
junctions or in gap junctions of other cell types. Because both neurons
and glia also are reported to express Cx32 (Nadarajah et al., 1996;
Dermietzel, 1998; Dermietzel and Spray, 1998; Alvarez-Maubecin et al.,
2000), we tested seven anti-Cx32 antibodies by FRIL. However, for
comparison with the antibodies most commonly used by other
investigators, images of Cx32 labeling by LM and FRIL are shown only
from samples labeled with monoclonal antibodies 7C7 and 2C2. The
specificities of these latter two antibodies have been documented (Li
et al., 1997). In addition, the usefulness of a newly developed
monoclonal anti-Cx30 antibody (33-2500; Zymed, San Francisco,
CA) is demonstrated.
Western blots. Male Sprague Dawley rats (300-350 gm) were
decapitated, and brain regions were dissected on ice and stored at
 80°C until use. Tissues were homogenized in ice-cold 40 mM Tris-HCl buffer, pH 7.4, containing 1% NP-40 detergent,
1 mM phenylmethylsulfonyl fluoride (PMSF), and aprotinin,
leupeptin, and pepstatin A each at 5 µg/ml. For tissues used in
immunoblots probed with anti-Cx43 antibody, homogenization buffer was
further supplemented with the phosphatase inhibitors sodium
orthovanadate and sodium fluoride at 1 and 10 mM
concentrations, respectively. After homogenization, samples were
sonicated for 20 sec. Total protein was determined with the Bio-Rad
(Hercules, CA) DC protein assay. Proteins were resolved by
SDS-PAGE, and gel percentage was varied according to the connexin to be
detected by immunoblotting (9% gels for Cx43, 12.5% gels for Cx30,
and 15% gels for Cx32). Before being loaded onto gels, samples probed
for Cx43 were boiled in sample buffer, whereas those probed for Cx30 or
Cx32 were applied without boiling. Resolved proteins were transferred
to 0.2 µm polyvinylidene difluoride membranes (Bio-Rad) in transfer
buffer [25 mM Tris, 192 mM glycine, and 20%
(v/v) methanol] containing 0.05% SDS. Membranes were blocked for 2-3
hr at 22°C in 20 mM Tris, pH 7.4, 150 mM
NaCl, and 0.2% Tween 20 (TBS-T) containing 5% skim milk powder and
incubated with primary antibody for 12-16 hr at 4°C in TBS-T
containing 1% skim milk powder. All primary antibodies were used at a
concentration of 1 µg/ml, except anti-Cx32 (Sigma, St. Louis, MO),
which was used at 0.33 µg/ml, and anti-Cx43 (18A), which was used at
a dilution of 1:35,000. After incubation with primary antibody,
membranes were washed for 40 min in TBS-T and then incubated for 1 hr
at room temperature in TBS-T containing 1% skim milk powder and either
anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary
antibody at dilutions of 1:5000 or 1:3000, respectively. Blots were
washed in TBS-T and then incubated for 1 min with ECL chemiluminescence
reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunohistochemistry for light microscopy. A newly generated
monoclonal anti-Cx30 antibody (Zymed 33-2500) was initially tested for
use in immunohistochemistry by peroxidase anti-peroxidase methods used
previously in studies of a polyclonal Cx30 antibody (Nagy et al.,
1999). For double-immunofluorescence studies, rats were deeply
anesthetized with equithesin and perfused transcardially with 50 ml of
prefixative solution consisting of cold (4°C) 0.1 M
sodium phosphate buffer (PB), pH 7.4, containing 0.9% saline (PBS) and
0.1% sodium nitrite and heparin (1 U/ml). For studies involving Cx30
and Cx43, rats were perfused with 400 ml of cold 4% formaldehyde in
PB, followed by postfixation of brains for 2 hr in the same fixative.
Tissues were stored at 4°C for 24-48 hr in cryoprotectant consisting
of 50 mM PB containing 10% sucrose. For studies involving
Cx43 and Cx32, rats were perfused with prefixative as above and then
with 400 ml of cold 4% formaldehyde in PB, followed by perfusion with
300 ml of 0.1 M PB containing 10% sucrose. Brains were
stored in cryoprotectant as above.
Cryostat sections (15 µm thick) were collected on gelatinized glass
slides. For Cx30/Cx43 double-immunofluorescence labeling, sections were
incubated for 24 hr at 4°C simultaneously with polyclonal rabbit
anti-Cx43 (18A) diluted 1:1000 and monoclonal mouse anti-Cx30 diluted
1:500 in PBS containing 0.3% Triton X-100 (PBST) and 2% normal goat
serum (NGS). For Cx43/Cx32 double-immunofluorescence labeling, sections
were incubated for 24 hr at 4°C simultaneously with polyclonal rabbit
anti-Cx43 (18A) diluted 1:1000 and monoclonal mouse anti-Cx32 (7C7)
diluted 1:25 in PBST containing 2% NGS. After primary antibody
incubations, sections were washed for 1 hr in PBST and then incubated
for 1.5 hr at room temperature with indocarbocyanine (Cy3)-conjugated
goat anti-mouse IgG (diluted 1:200 in PBST containing 2% NGS) for
labeling of either Cx30 or Cx32 or, simultaneously, with fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (diluted 1:50 in
PBST containing 2% NGS) for labeling of Cx43. Sections were washed for
20 min in PBST and 20 min in 50 mM Tris-HCl buffer and
coverslipped with anti-fade medium. Immunofluorescence double labeling
for Cx32 and CNPase was conducted using monoclonal anti-Cx32 7C7 and a
polyclonal anti-CNPase as described previously (Li et al., 1997). In
control procedures, omission of one or the other of the primary
antibodies with inclusion of both of the secondary antibodies produced
no inappropriate labeling (i.e., Cy3 labeling with rabbit primary or
FITC labeling with monoclonal primary), indicating a lack of false-positive cross-reactions with the antibodies used. In addition, adsorption of the above anti-connexin antibodies with peptide antigen
has been shown previously to eliminate all immunolabeling in tissue
sections (Yamamoto et al., 1990b; Li et al., 1997; Nagy et al.,
1999).
Fluorescence was examined on a Leitz Dialux 20 fluorescence microscope
and an Olympus Fluoview confocal microscope. By the use of tissue
sections singly labeled with either FITC- or Cy3-conjugated antibodies,
confocal laser excitation intensity and photomultiplier tube detection
of these labels were adjusted to produce minimal bleedover of the green
fluorochrome into the red range. This was necessary because of the
extended tail of long-wavelength emission of FITC, even with the use of
appropriate 605-610 nm cutoff filters, as used here. Lack of bleedover
was tested by scanning double-labeled sections twice using single laser
excitation each time for one or the other fluorochrome.
Freeze-fracture. For FRIL immunocytochemistry, adult
Sprague Dawley rats (11 males and 14 females; 128-585 gm) were
anesthetized (ketamine, 90 mg/kg; xylazine, 8 mg/kg) and fixed for
3-10 min via transcardiac perfusion with 4, 1, 0.2, or 0.1%
formaldehyde in 150 mM Sorenson's phosphate buffer (SPB)
as described previously (Hudson et al., 1981). Optimum cell
preservation and labeling for connexins were obtained after fixation
with 1% formaldehyde, and all images are from that procedure (for
exceptions, see Figs. 5, 7F, which were from tissues
fixed with 0.1% formaldehyde). All experiments were conducted
according to the Principles of Laboratory Animal Care
(National Institutes of Health publication number 86-23; revised 1985).
Samples of formaldehyde-fixed brain and spinal cord were cut into
150-µm-thick slices using a Lancer 1000 Vibratome, infiltrated with
30% glycerol, and frozen by contact with a 195°C "copper
mirror" (Phillips and Boyne, 1984). Samples were freeze fractured in
a JEOL RFD 9010C freeze-fracture device, shadowed with 1-1.5 nm of
Pt/C, and coated with 5-10 nm of carbon. Frozen samples were
bonded to gold "index" grids using 1.5-2% Lexan plastic (GE
Plastics, Pittsfield, MA; available locally from plastic sheet-goods
suppliers) dissolved in dichloroethane and photography-mapped using a
Molecular Dynamics MultiProbe 2001 inverted confocal microscope (Rash
et al., 1995, 1997). Replicas were washed in 2.5% SDS detergent for
24-29 hr with constant stirring, which leaves a thin film of membrane
macromolecules adsorbed to the replica and available for immunogold
labeling (Fujimoto, 1995; as modified in Rash and Yasumura, 1999).
Blocking nonspecific binding sites; immunogold labeling.
Samples were immersed for 1-1.5 hr at 22-24°C in primary antibody solution (1 mg/ml stock solution of single antibodies or mixed primary
antibodies from two or three species) mixed 1:100 with labeling
blocking buffer (Rash et al., 1999), which consists of 0.15 M SPB plus 10% heat-inactivated goat serum and 0.5%
teleost gelatin (Sigma). Two anti-Cx30, seven anti-Cx32, two anti-Cx36, and three anti-Cx43 antibodies were used, details of which are given in
Tables 1 and 2. In some double- and triple-labeling experiments, the
identity of astrocyte processes was confirmed by labeling the square
array markers of astrocytes (AQP4 arrays) with anti-AQP4 antibodies
(Rash et al., 1998b; Rash and Yasumura, 1999). Replicas were labeled
for 1.5 or 12 hr with species-specific secondary antibodies (goat
anti-mouse, goat anti-rabbit, and donkey anti-sheep) coupled to 10 nm,
20 nm, or 30-40 nm gold (Chemicon, Temecula, CA; now available
directly from Jackson ImmunoResearch, West Grove, PA, and NanoProbes,
Stony Brook, NY).
Electron microscopy. Labeled replicas were rinsed and air
dried, and a second reinforcing carbon coat was applied on the labeled "tissue side" of the replica. The second carbon coat prevented displacement of the replica and of the gold labels during subsequent removal of the Lexan support film (Rash et al., 1995; Rash and Yasumura, 1999). The Lexan support film was removed by immersing the
grid in six exchanges of dichloroethane for a total of 1-1.5 hr. Grids
were air dried and examined at 100 kV using a JEOL 2000 EX-II TEM.
Overall, 303 labeled replicas were examined (Table 2) and photographed
as stereoscopic pairs, with an 8° included angle between images. Most
images were printed at low and high magnification to allow assessment
of the level of background immunogold labeling, which is always present
with any particulate (i.e., immunogold) labeling procedure, even if not
specified. When nonspecific labeling is high, discriminating signal
from background is difficult, if not impossible. Consequently, the
amount of clumping and of nonspecific labeling, as well as the
"signal-to-noise ratio" (Rash and Yasumura, 1999), were determined
for each replica that was used for quantitative analysis. Gold beads
closer than 30 nm apart were considered to represent a single
label. Omission of primary or secondary antibody resulted in the
absence of labeling for that connexin (negative control). Many samples
were examined and photographed "blind" with respect to each
antibody label. Approximately half of the replicas were examined only
briefly because of high background (potentially producing
false-positive labeling), failure of secondary labels (yielding false
negatives), clumping of labels, or other artifacts of labeling (Rash
and Yasumura, 1999). The total area per replica occupied by
oligodendrocyte and neuronal gap junctions was small (<1
µm2 of gap junction per 100,000 µm2 of replica surface), and the
nonspecific background was <1 gold bead/µm2. Consequently, the likelihood
of finding nonspecific gold beads at gap junctions of oligodendrocytes
and neurons was low, and false-positive labeling was a negligible
factor in the analysis of cell coupling partners by FRIL. Furthermore,
>90% of nonspecific labels were on the Lexan (nontissue) side of the
replica, allowing most nonspecific labeling to be distinguished from
specific labeling (for rationale, see Rash and Yasumura, 1999).
Stereoscopic images are presented to allow confirmation that all
specific immunogold labeling was on the cytoplasmic side of the
replicated gap junctions. (Stereoscopic images should be viewed with a
conventional prop-up or stereopticon-type viewer.) In contrast, several
samples were found to have no labeling of known connexin components in
any gap junctions of the appropriate cell type (false-negative
labeling). False-negative labeling was usually traced to bad lots of
primary or secondary antibodies, and those replicas were either
discarded (single labels), or if two or three labels were used, data
were obtained only for the other labels (i.e., each label was evaluated independently of any other label that might be present). For FRIL, each
lot of primary and secondary antibodies was tested and discarded when
it had exceeded its useful shelf life (<6 months at 4°C for both
primary and secondary antibodies). Primary and secondary antibodies
were not refrozen because each freeze-thaw cycle resulted in increased
clumping of immunogold beads.
For the series of replicas used for quantitative FRIL analysis of
oligodendrocyte coupling partners, oligodendrocyte extraplasmic leaflets were examined at high magnification for the presence of gap
junctions. Counting of labeled versus unlabeled gap junctions was
performed on oligodendrocyte coupling partners in the suprachiasmatic nucleus, hippocampus, and spinal cord. Nonquantified but directly supporting data were also obtained from gray matter areas of the paraventricular nucleus, cerebellum, and supraoptic nucleus. For quantification of Cx36-labeled neuronal gap junctions, samples of
retina, inferior olive, and spinal cord were examined. To minimize bias
in data collection and interpretation in samples used for quantitative
analysis of cell coupling partners, every oligodendrocytic or neuronal
membrane with either labeled or unlabeled gap junctions was
photographed. No attempt was made to quantify connexin proteins; only
the number of gap junction plaques containing gold labels for each
connexin type was determined. In replicas used for quantitative analysis of cell coupling partners, the signal-to-noise ratio [defined as the density of gold beads per unit area of gap junction divided by the density of gold beads on nonjunctional areas of the same
replica (Rash and Yasumura, 1999)] varied from 500:1 to 5000:1. In
most samples, the labeling efficiency was  1 gold bead per 30 connexons, which is sufficient to result in multiple gold beads (as
many as 150) on all except the smallest gap junctions. In optimum
conditions, each gold bead acts as an independently targeted label.
Thus, multiple gold beads on an individual gap junction provide
multiple independent confirmations of the presence of the target
connexin within that specific gap junction plaque. In addition,
consistent labeling for the same connexin in multiple gap junctions on
multiple cells of a single cell type provides additional confidence of
labeling efficacy and precludes "false-positive" assignment to
inappropriate cells or inappropriate membrane appositions. For each of
the four connexins tested, this level of labeling was sufficient to
label >90% of gap junctions in each cell type and to label >98% of
gap junctions containing >150 connexons. In most cases, cell-specific
ultrastructural markers and immunocytochemical labeling were used to
confirm cell identifications (Rash et al., 1997).
| |
RESULTS |
Western blotting
Western blot analyses of nine of the anti-connexin antibodies used
in the present study are shown in Figure
1. As reported previously in studies
involving CNS tissues (Nagy et al., 1999), polyclonal anti-Cx30
antibody 71-2200 detected a single band migrating at ~30 kDa (Fig.
1A, lane 1), and monoclonal anti-Cx30
detected a similar band corresponding to Cx30 (Fig.
1A, lane 2). The monoclonal antibody also
detected higher molecular weight bands that may correspond to
multimeric forms of Cx30. The extensively used polyclonal anti-Cx43
antibody 18A recognized Cx43 migrating at ~43 kDa (Fig. 1B, lane 1), and the commercially
available monoclonal anti-Cx43 used here reacted with a corresponding
protein (Fig. 1B, lane 2). The anti-Cx32
antibody 7C7 detected Cx32 monomer as well as its higher molecular
weight dimer form in homogenates of liver and spinal cord (Fig.
1C, lanes 1, 2, respectively).
Corresponding bands were revealed by each of the other anti-Cx32
antibodies tested in homogenates of spinal cord (Fig. 1C,
lanes 3-6). Additional lower molecular weight
proteins of unknown identity were detected by two of the commercially
available anti-Cx32 antibodies (Fig. 1C, lanes 4, 5).
View larger version (36K):
[in this window]
[in a new window]
|
Figure 1.
Western blots showing connexin detection in the
CNS with the various anti-connexin antibodies used for LM
immunofluorescence and FRIL. A, Blots of whole-brain
homogenate probed with polyclonal (lane 1) and
monoclonal (lane 2) anti-Cx30 antibodies. The top
two intense bands detected by the monoclonal antibody may
correspond to Cx30 dimer and trimer forms. B, Blots of
whole-brain homogenate probed with polyclonal (lane 1)
and monoclonal (lane 2) anti-Cx43 antibodies.
C, Blots of liver and spinal cord homogenates probed
with anti-Cx32 antibodies. Lanes 1, 2, Monoclonal 7C7;
lane 3, Zymed monoclonal 2C2; lane 4,
Sigma polyclonal; lane 5, Chemicon polyclonal;
lane 6, Zymed polyclonal. Each of the antibodies detects
the monomer form of Cx32 and to varying degrees its dimer form. The
Sigma and Chemicon antibodies detect a lower molecular weight band of
uncertain identity. Numbers on the left of each
blot correspond to molecular weight markers.
|
|
LM immunohistochemistry
The general appearance of immunolabeling produced with anti-Cx30,
anti-Cx43, and anti-Cx32 antibodies at low and medium magnification of
brain sections is shown in Figure 2.
Results with monoclonal anti-Cx30 were qualitatively similar to those
reported previously using a polyclonal antibody against Cx30 (Nagy et
al., 1999). Labeling for Cx30 was detected throughout the brain, but
its distribution was highly heterogenous in gray matter, and it was not
detectable in white matter tracts. For example, in a representative
section through portions of the basal ganglia, punctate
immunoperoxidase staining for Cx30 was very dense in the globus
pallidus and moderate to weak in the striatum (Fig.
2A), presumably reflecting differences in the number
of astrocytes and/or astrocytic gap junctions in those areas.
View larger version (123K):
[in this window]
[in a new window]
|
Figure 2.
Photomicrographs showing patterns of
immunolabeling obtained with various anti-connexin antibodies used in
double-immunofluorescence and FRIL studies. A, Low
magnification of immunoperoxidase labeling (arrows) with
a monoclonal anti-Cx30 antibody. The section shows dense punctate
labeling in the globus pallidus (GP) and weaker labeling
in the striatum (St). B, Cerebral cortex
showing a high concentration of punctate immunofluorescence with
anti-Cx43 antibody 18A. Dark ovals represent unstained
neuronal cell bodies. C, Cx32 labeling of myelinated
fibers (arrows) as well as oligodendrocyte cell bodies
(arrowheads; cells barely discernable at this
magnification) using antibody 7C7. D, E, Higher
magnifications showing Cx32-positive puncta with antibody 7C7
along the surface of oligodendrocytes in the cerebral cortex
(D; arrows) and ventral lateral nucleus of the thalamus
(E; arrows). F,
Through-focus confocal micrograph of Cx32-immunopositive puncta
distributed on an oligodendrocyte soma (arrows) and its
processes (arrowheads). G, Double
immunofluorescence of the oligodendrocyte marker CNPase
(G1, green) and Cx32 (G2,
red) in the same field of cerebral cortex. CNPase-positive
cells are also immunopositive for Cx32 as seen by the
overlay of images (G3,
yellow). H, Higher magnification
double-immunofluorescence confocal micrographs showing a
CNPase-positive oligodendrocyte in cerebral cortex (H1)
decorated with numerous Cx32-immunopositive puncta (red
puncta in H2 and red and
yellow puncta in the overlay of images in
H3). Scale bars: A, 200 µm; B,
C, 50 µm; D, E, G, 20 µm; F,
H, 5 µm.
|
|
Characteristic immunofluorescence-labeling patterns obtained with
anti-Cx43 and anti-Cx32 antibodies are shown at low magnification in
images from cerebral cortex labeled with antibody 18A (Fig. 2B) and 7C7 (Fig. 2C), respectively.
Labeling for Cx43 was dense, widely distributed in both gray and white
matter, and exclusively punctate, whereas labeling for Cx32 was seen
along myelinated fibers and around numerous cells that were identified
as oligodendrocytes on the basis of double labeling for Cx32 in
combination with oligodendrocyte ultrastructural and cytochemical
markers (Li et al., 1997) (see images below). Although visualization of
immunopositive oligodendrocyte somata was obscured at low magnification
by labeled myelinated fibers (Fig. 2C), oligodendrocytes
were evident in magnified images, in which their cell bodies and
initial processes were delineated by punctate immunofluorescence, as
shown by conventional LM of cerebral cortex (Fig. 2D)
and the ventral lateral nucleus of the thalamus (Fig.
2E). By high-magnification through-focus confocal microscopy of a single oligodendrocyte in the hippocampus (Fig. 2F), >60 puncta were present on the oligodendrocyte
cell body and proximal processes. Confirmation that these puncta were
associated with oligodendrocytes was obtained in numerous brain areas
double-labeled for Cx32 and an oligodendrocyte-specific marker, CNPase.
Examples are shown by standard fluorescence (Fig. 2G) and
confocal (Fig. 2H) microscopy in areas of cerebral
cortex. CNPase was localized to oligodendrocyte somata and their
myelinating processes along axons (Fig. 2G1), both
of which also were immunopositive for Cx32 (Fig.
2G2,G3). Within this meshwork of labeling, CNPase-positive oligodendrocyte cell bodies, and occasionally their initial processes, were consistently outlined by numerous Cx32-immunopositive puncta (Fig.
2G2,G3). Where CNPase was not present, labeling for Cx32 was
not detected, suggesting that Cx32 is present only in oligodendrocytes.
Double immunofluorescence for glial connexins, as visualized by
confocal microscope scans of 1-3 µm optical slices of tissue, is
shown in Figure 3. In sections
double-labeled for Cx43 and Cx30, correspondence of punctate
immunofluorescence in gray matter was high in each of several brain
regions examined. This was exemplified in images of the subthalamic
nucleus in which dense punctate staining for Cx43 (Fig.
3A2) and Cx30 (Fig. 3A1) was often closely
associated (Fig. 3A3). (We use "close association"
rather than "colocalization" because the resolution of confocal
microscopy cannot distinguish between structures separated by <0.3
µm.) In these double-labeled sections, Cx43 was evident in white
matter, whereas no labeling for Cx30 was detected in white matter
tracts, such as the corpus callosum, anterior commissure, and internal
capsule. In sections labeled for both Cx43 and Cx32, a small proportion
of Cx43-immunopositive puncta in gray as well as white matter was
associated with oligodendrocyte cell bodies. Among the numerous
Cx43-positive puncta (Fig. 3B1,C1,D1, green
immunofluorescence), a small fraction were closely associated with
Cx32-positive puncta (Fig. 3B2,C2,D2, red
immunofluorescence), which delineated oligodendrocyte cell
bodies. This close association of Cx43 and Cx32 (double
immunofluorescence appearing yellow) is evident in overlays
of corresponding images taken in regions of cerebral cortex (Fig.
3B3), the ventral nucleus of the thalamus (Fig.
3C3), and the lateral hypothalamus (Fig. 3D3).
Close association of Cx32 and Cx43 was evident at hundreds of
oligodendrocytes that were examined in dozens of brain regions in
sections derived from several animals. Overall, however,
Cx43-immunopositive puncta greatly outnumbered (>100:1) the Cx32- plus
Cx43 double-labeled puncta surrounding individual oligodendrocytes. A
basis for the close association of Cx32 and Cx43 puncta was ascertained
by FRIL (below).
View larger version (86K):
[in this window]
[in a new window]
|
Figure 3.
Confocal microscope images showing
double-immunofluorescence labeling for connexins in various brain
regions. A1-A3, Labeling for Cx30 (A1)
and Cx43 (A2) in the subthalamic nucleus with the
overlay (A3) showing colocalization in
yellow. B-D, Labeling for Cx43
(green in B1, C1, D1) and Cx32
(red in B2, C2, D2) with the
overlay showing colocalization
(yellow in B3, C3, D3) of
immunofluorescent puncta surrounding an oligodendrocyte (OL,
arrow) in the cerebral cortex (B), the
ventral lateral thalamic nucleus (C), and the
lateral hypothalamus (D). Scale bars, 5 µm.
|
|
Freeze-fracture immunogold labeling
FRIL provides for immunocytochemical identification of
membrane proteins and subcellular localization to distinct
ultrastructural features, such as gap junctions (Fig.
4). In FRIL, the fracture plane may split
either of the apposed membranes or sequentially split both apposed
membranes, revealing the protoplasmic leaflet (P-face) of the lower
cell, composite P-face and extraplasmic leaflet (E-face) images derived
from portions of both cells (Fig. 4B,E), or only the
E-face of the upper cell (Fig. 4F). Although membranes are split, the fracture plane does not break covalent bonds
within transmembrane proteins but, instead, separates the apposed
connexons at the point of apposition in the extracellular space. Thus,
in vertebrates, each gap junction is cleanly separated into two plaques
of hemichannels, with each plaque containing connexins derived from
only one cell (Fig. 4, orange vs yellow connexons). In P-faces, connexons are replicated as distinctive arrays
of intramembrane particles (IMPs). The removal of connexons by cleaving
leaves equally distinctive arrays of impressions or "pits" in the
E-face, which is devoid of connexin proteins. However, every gap
junction E-face overlies the unsplit gap junctional membrane of the
lower cell, which contains the connexons and connexin proteins of the
lower cell (Fig. 4C,F). Thus, whether the fracture plane exposes the E-face of the upper cell, exposes the P-face of the
lower cell, or repeatedly steps from E- face to P-face within a gap
junction, it is only the connexons of the lower cell that remain for
immunogold labeling (Fig. 4C-F, yellow
connexons). Moreover, no contaminating connexins from the upper cell
remain in the replicated E-face and, thus, cannot cause false-positive FRIL labeling of connexins in the lower cell. [This report does not
illustrate labeling of cross-fractured gap junctions or areas where the
cleaving plane exits the membrane and enters the cytoplasm of the upper
cell. Those sources of "cryptic labeling" are described in Rash and
Yasumura (1999).] Consequently, in addition to its ability to identify
connexins in gap junction P-faces (Fig. 4D), a unique
advantage of FRIL in this investigation is its ability to allow
visualization and positive structural identification of the external
leaflet (E-face) of one cell while revealing the connexin composition
of the attached cellular coupling partner (Fig. 4E).
Thus, for classes of connexins that are restricted to a specific cell
type (as documented below), FRIL provides a method to identify the
lower cell that is based solely on its constituent connexins, even when
little or none of the P-face or cytoplasm of the lower cell is visible
in the replica (Fig. 4F). However, in most cases,
portions of both cells were replicated, allowing other ultrastructural
features to be used to confirm cell identifications. This cell-specific
connexin labeling within individual gap junction plaques provides the
basis for FRIL identification and quantitative analysis of
oligodendrocyte and neuronal coupling partners. Whether or not
additional connexins are found to be shared among glial cells and/or
neurons, the cell-specific expression of these four connexins allows
each to be used as a marker for identifying cellular coupling
partners.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 4.
Diagrammatic representation of FRIL showing
relationships between visualized freeze-fracture faces and the
location of immunogold-labeled connexins. A, A gap
junction between two cells is shown where the fracture plane
(blue dashed line) skips from the membrane bilayer of
one cell to that of the other cell but always separates apposed
connexons at the point of contact in the extracellular space. B,
C, Where the membrane of the upper cell is fractured, the
external leaflet (E-face) remains attached to the tissue fragment that
contains the lower cell. The E-face pits delineate the sites from which
the connexons were removed. Curved arrow indicates
separation and removal of upper fragment. Pt arrow indicates
direction of platinum shadowing. D-F, Where the
fracture plane splits the membrane of the lower cell, the external
leaflet is removed, exposing connexons as intramembrane particles in
the protoplasmic leaflet (P-face). In all cases, regardless of whether
the E-face of the upper cell or the P-face of the lower cell is
replicated, only the connexons of the lower cell remain for potential
labeling by FRIL.
|
|
Cx32 at oligodendrocyte gap junctions but not in
oligodendrocyte coupling partners
Oligodendrocytes were identified in freeze-fracture
replicas according to established criteria, including the following:
(1) characteristic low density of IMPs in both P- and E-faces,
particularly in myelin (see Figs. 5D, 7, 9), (2) the
presence of "reciprocal patches" of mixed IMPs and pits in both E-
and P-faces (see Figs. 5, 8), and (3) noncrystalline packing of IMPs
and pits in their gap junctions (see Figs. 5, 7-9) (Hatton and
Ellisman, 1981; Landis, 1981; Massa and Mugnaini, 1982; Waxman and
Black, 1984; Mugnaini, 1986; Rash et al., 1997). Likewise, astrocytes
and neurons were identified on the basis of published criteria (Hatton
and Ellisman, 1981; Landis, 1981; Mugnaini, 1986; Rash et al., 1996,
1997), as described below. In freeze-fracture replicas of spinal cord, hippocampus, suprachiasmatic nucleus, paraventricular nucleus, and
cerebellum that were examined after single, double, or triple labeling
for various combinations of Cx32 plus Cx30, Cx36, Cx43, and/or AQP4
(Table 2), as many as 41 intercellular gap junctions were observed on
P-faces of individual oligodendrocyte somata, in this case, 35 of which
were labeled for Cx32. In samples with high labeling efficiency, 83 of
98 gap junctions in oligodendrocyte P-faces were labeled for Cx32
(Table 3). Of these, 48 labeled gap
junctions were in somatic plasma membranes and 35 were in myelin (see
below). In several instances in which sufficient area was present to
permit positive identification of both cells, all coupling partners
overlying oligodendrocyte P-faces were identified as astrocytes, and
the oligodendrocyte sides of these astrocyte-to-oligodendrocyte junctions were consistently labeled for Cx32 (Fig.
5B). Cx32-labeled oligodendrocyte gap junctions consisting of 9-1300 connexons were labeled by 1-31 gold beads (range illustrated in Fig.
5B-D). This overall labeling efficiency of 1 gold per 30 connexons (1:30), combined with low nonspecific background and high
signal-to-noise ratio, resulted in multiple labeling of all except the
smallest gap junctions (i.e., <30 connexons).
View this table:
[in this window]
[in a new window]
|
Table 3.
Summary of cellular coupling relationships and FRIL-labeled
connexin constituents of gap junctions between cell types
|
|
View larger version (147K):
[in this window]
[in a new window]
|
Figure 5.
Stereoscopic images of Cx32 labeling of gap
junctions in oligodendrocyte plasma membranes in the cerebellum, with
explanatory diagrams. A, Oligodendrocyte cell body with
a broad expanse of plasma membrane P-face (P)
enclosing the cross-fractured cytoplasm (asterisk).
Arrows point to two Cx32-labeled gap junctions in the
oligodendrocyte P-face (white arrows) and one unlabeled
gap junction in the nearby astrocyte P-face (black arrow). An additional gap
junction with ~280 connexons was labeled by 12 gold beads
(boxed area; enlarged in B). On the
right of each stereo pair is an interpretive
drawing, in which each cell type is delineated by
different shading and a stylized complement of IMPs or
pits in each membrane fracture face is shown. B,
Cx32-labeled gap junction linking a confirmed astrocyte finger that was
fractured from its P-face (As P), through its cytoplasm
(asterisk), to its E-face (As E). Air
drying of the labeled replicas resulted in collapse of the labels to
the underside of the replica. Ol P, oligodendrocyte
P-face. C, Cx32-labeled gap junction consisting of 1280 connexons that was labeled by 30 gold beads (black
dots). A nearby gap junction consisting of <40 IMPs was
unlabeled. The reciprocal patch (arrow) and paucity of
IMPs are characteristic of oligodendrocyte P-faces. D,
Cx32-labeled gap junction (enlarged in inset) in the
P-face of the outer layer of myelin, directly adjacent to the outer
tongue (OT). The outer tongue of myelin
terminates at a tight junction consisting of several rows of P-face
furrows (arrow) and IMP ridges. Three gold beads were
bound to the oligodendrocyte side of the gap junction. In all FRIL
images (in this and subsequent figures), scale bars are 0.1 µm unless
otherwise indicated.
|
|
Oligodendrocyte gap junctions were also observed in the outermost layer
of myelin, where only oligodendrocyte-to-astrocyte gap junctions have
been documented (Waxman and Black, 1984; Mugnaini, 1986; Rash et al.,
1997, 1998b). Even in gap junctions having few connexons, the
oligodendrocyte sides of intercellular gap junctions in the outer
surface of myelin were consistently labeled for Cx32 (Fig.
5D). Overall, 35 of 44 intercellular gap junctions in the outermost, uncompacted layer of
myelin were labeled for Cx32. [This study of cellular coupling
partners did not address the composition or distribution of autologous
or "reflexive" gap junctions linking layers deeper within myelin
(Sandri et al., 1977; Scherer et al., 1995).] In contrast to gap
junctions in oligodendrocyte P-faces, nearby astrocyte P-faces (Fig.
5A, black arrow) and neuronal P- and E-faces were
never labeled for Cx32. The absence of Cx32 in neurons is particularly
noteworthy because in ~80 of the Cx32-labeled replicas of brain and
spinal cord, extensive searches had been conducted using Cx32
immunogold as "flags" to aid in searches for proposed
Cx32-containing neuronal gap junctions (Micevych and
Abelson, 1991; Nadarajah et al., 1996; Alvarez-Maubecin et al., 2000).
However, in tissues in which we had previously found >100 neuronal gap
junctions (Rash et al., 1996, 2000), no Cx32-labeled neuronal gap
junctions were found. Overall, Cx32 was detected by FRIL only in gap
junction plaques of oligodendrocyte plasma membranes and never in
astrocytes or neurons (Table 3).
View larger version (132K):
[in this window]
[in a new window]
|
Figure 6.
Stereoscopic images of astrocyte gap
junctions in the suprachiasmatic nucleus after labeling for Cx43 and
AQP4 (A), in the supraoptic nucleus after
labeling for Cx30 (B), in the supraoptic nucleus
after double labeling for Cx43 and Cx30 (C), and
in the paraventricular nucleus after triple labeling for Cx30, Cx32,
and Cx43 (D). A, Two
astrocyte-to-astrocyte gap junctions labeled for Cx43 (20 nm beads;
large arrows) beneath their E-faces
(E) and P-faces (P). Square
arrays in P-faces were labeled for AQP4 (10 nm gold beads; small
arrow and inset). B,
Astrocyte-to-astrocyte gap junction labeled for Cx30 (10 nm gold).
C, A gap junction linking two astrocyte processes after
double labeling for Cx30 (10 nm gold beads) and Cx43 (20 nm gold
beads). Astrocytes were positively identified by the presence of AQP4
square arrays in both P-faces (white arrows) and E-faces
(black arrows), as well as by the high density of IMPs
on both P- and E-faces. D, Two astrocyte gap junctions
triple-labeled for Cx30 (20 nm gold), Cx43 (30-40 nm gold), and Cx32
(10 nm gold; none present). Cx32 was not detected in
astrocyte-to-astrocyte gap junctions but was detected in
astrocyte-to-oligodendrocyte gap junctions (Fig. 5).
|
|
Although gap junctions were consistently labeled for Cx32 in
oligodendrocyte plasma membrane P-faces (Table 3), no connexins were
labeled for Cx32 in the cells beneath oligodendrocyte gap junction
E-faces (i.e., in oligodendrocyte coupling partners; Table 3; see Fig.
8C), suggesting that few if any oligodendrocytes share gap
junctions with other oligodendrocytes. Moreover, the absence of Cx32
labeling in the gap junctions of oligodendrocyte coupling partners
could not be explained by increased susceptibility to SDS detergent
solubilization of oligodendrocyte connexins beneath E-faces because
Cx32 labeling was frequently found in oligodendrocyte plasma membranes
beneath astrocyte E-faces at confirmed
astrocyte-to-oligodendrocyte gap junctions (Fig. 5B). Thus,
the absence of Cx32 in oligodendrocyte coupling partners required that
additional samples be examined by FRIL using antibodies to Cx30, Cx43,
and Cx36, which are connexins expressed in the other cell types with
which oligodendrocytes have been proposed to couple (Massa and
Mugnaini, 1982; Mugnaini, 1986; Nadarajah et al., 1996; Rash et al.,
1997; Alvarez-Maubecin et al., 2000).
Cx43 and Cx30 as definitive markers of astrocyte
gap junctions
To investigate Cx43 localization by FRIL, particularly with
respect to oligodendrocyte coupling partners, 126 replicas of adult rat
brain and spinal cord were single-, double-, or triple-labeled with
antibodies to Cx43 plus various combinations of consensus neuronal and
glial connexins (Cx30, Cx32, and Cx36), with some replicas also labeled
for AQP4 to confirm astrocyte identities. In neuropil, >1500
Cx43-labeled gap junction P-faces were found, and these were always in
astrocytes (Table 3; Fig. 6A) or in ependymocytes
(Rash et al., 1998b; Rash and Yasumura, 1999). In addition, >1500
Cx43-labeled astrocyte gap junction E-faces were found, >95% of which
were between two astrocytes. (The remaining astrocyte coupling partners
were oligodendrocytes; see below.) Confirmation that the cells forming
these Cx43-labeled gap junctions were astrocytes was based on both
ultrastructural and biochemical markers, including the following: (1)
the presence of labeled or unlabeled AQP4 arrays in their plasma
membranes (Fig. 6A,B; also see Fig. 5B),
(2) a distinctively high density of IMPs in their plasma membrane E-
and P-faces (Fig. 6A,C; also see Fig. 5B),
and (3) clusters of GFAP filaments in their cross-fractured cytoplasms
(see Fig. 8A,B). In one series of samples that had very low background and high signal-to-noise ratio, ~90% of
astrocyte gap junctions were labeled for Cx43, and the remaining 10%
(mostly gap junctions with <50 connexons) were unlabeled. Moreover,
Cx43 was not detected in the plasma membranes of any other cell type in
gray or white matter. [FRIL analysis of ependymocyte gap junctions revealed that they also contain Cx43 (Rash et al., 1998a,b; Rash and
Yasumura, 1999), but because ependymocytes are restricted to the
linings of the brain ventricles and spinal cord central canal, they
were excluded from this analysis of neuronal and glial coupling
partners in neuropil and white matter.]
In samples labeled for Cx30 (see Figs. 6B,
8C) or Cx30 plus Cx43 (see Figs. 6C,D,
9B), >200 astrocyte gap junction plaques were found,
~90% of which were labeled (Table 3). By alternately using large
versus small gold beads targeted to Cx43 and Cx30, approximately equal
amounts of Cx30 and Cx43 were detected in most of the larger
astrocyte-to-astrocyte gap junctions (Fig. 7C). To test whether the high
proportions of gap junctions that were separately labeled for Cx43 or
Cx30 reflected an artifact of a selective search strategy (i.e., biased
searching only for labeled gap junctions) or accurately reflected
overlapping populations of gap junctions expressing two or more
connexins, labeled and unlabeled gap junctions were quantified after
simultaneous double labeling for Cx30 and Cx43 (with or without
additional labeling for Cx32 or Cx36). Labels for both Cx30 and Cx43
were present in ~80% of gap junctions in astrocyte plasma membranes
(Fig. 6B,C). Nevertheless, ~15% of small gap
junctions were labeled for only one of the two connexins, and ~5%
were unlabeled (Figs.
8A,
9B). In contrast, no gap
junctions in either oligodendrocyte or neuronal P-faces were labeled by
Cx43 or Cx30 (Table 3). Thus, of the connexins tested by FRIL, Cx30 and
Cx43 were colocalized only in the gap junction plaques of astrocytes
and thus are definitive FRIL markers of astrocytes and no other neural
cells.
View larger version (144K):
[in this window]
[in a new window]
|
Figure 7.
Stereoscopic images of paired oligodendrocytes
linked by tight junctions in the suprachiasmatic nucleus
(A-E) after labeling for Cx43 and AQP4 and in
the inferior olive (F) after double labeling for
Cx43 (10 nm gold) and Cx36 (20 nm gold; none present in any
oligodendrocyte gap junction). A, Low-magnification
image of the large expanse of oligodendrocyte concave plasma membrane
E-face contacting the cell body of a second oligodendrocyte, which has
cross-fractured cytoplasm (asterisk).
Boxes delineate areas of tight junctions
(B) and 2 of the 12 Cx43-labeled gap junctions
seen in this oligodendrocyte E-face (C).
B, Higher magnification image of the region linked by
tight junctions. No gap junctions were within the sealed compartments
enclosed by tight junction strands, but gap junctions were numerous in
the nearby plasma membrane. C, Higher magnification
image of two Cx43-labeled gap junctions whose connexons were in the
plasma membrane of the oligodendrocyte coupling partner (boxed
areas D, E, shown at higher magnification). D,
E, Gap junctions labeled for Cx43 by 10 nm gold. "Reciprocal
patches" of IMPs (arrows) were present in the margins
of both gap junctions. Cx36 labeling (20 nm gold) was not present in
any oligodendrocyte coupling partner. F, Tight junction
strands linking two oligodendrocytes, with nearby gap junctions in the
oligodendrocyte coupling partner labeled for Cx43.
|
|
View larger version (171K):
[in this window]
[in a new window]
|
Figure 8.
Identification of astrocytes as oligodendrocyte
coupling partners in suprachiasmatic nucleus (A, B) and
spinal cord (C). A, Stereoscopic
image of an oligodendrocyte linked to the same astrocyte by one
unlabeled gap junction (top left box, enlarged in
top right inset) and by one Cx43-labeled gap junction
(20 nm gold; bottom left box, enlarged in bottom
right inset). The coupled astrocyte contained a small bundle of
GFAP filaments in the cross-fractured cytoplasm
(asterisk), and a square array was labeled for AQP4 (10 nm gold beads; white arrow). In oligodendrocyte E-faces,
reciprocal patches (black arrow) contain both IMPs and
pits. B, Stereoscopic image of one Cx43-labeled gap
junction and one unlabeled mixed gap junction/reciprocal patch
(arrow) in an oligodendrocyte E-face. GFAP filaments are
in the cross-fractured cytoplasm (asterisk).
C, Stereoscopic image of a gap junction in a spinal cord
oligodendrocyte E-face in a sample that was double-labeled for Cx32 (10 nm gold; none present) and Cx30 (20 nm gold). Only Cx30 labeling was
present in the plasma membrane of the oligodendrocyte coupling partner.
Gap junctions often abut or intermingle with reciprocal patches
(arrow).
|
|
View larger version (164K):
[in this window]
[in a new window]
|
Figure 9.
Oligodendrocyte-to-astrocyte gap junctions
in myelin from suprachiasmatic nucleus after labeling for Cx43
(A) and in oligodendrocyte plasma membrane from
supraoptic nucleus after double labeling for Cx43 and Cx30
(B). A, Small gap junction on
outer myelin plasma membrane E-face labeled for Cx43 by four 20 nm gold
beads (enlarged in adjacent inset). Continuity
of the E-face (arrow labeled E) may be
traced from the outer layer of myelin (My) to the gap
junction. Outer myelin membrane had IMP-free areas and reciprocal
patches. Tight junction strands (white arrows) linked a
small patch of the second layer of myelin to the outer layer of myelin.
B, Four gap junctions (1-4) in
somatic plasma membrane of an oligodendrocyte after double labeling for
Cx43 (20 nm gold beads) and Cx30 (10 nm gold beads). Two small gap
junctions (2, 3) were not labeled, and
one (4) was labeled by only two 10 nm gold beads
(Cx30). Oligodendrocyte myelin E-faces are almost devoid of IMPs.
|
|
Oligodendrocyte coupling partners have Cx30 and Cx43 in
their plasma membranes
In replicas that were single-, double-, or triple-labeled
for Cx30 and Cx43 plus Cx32 or Cx36, the connexins beneath
oligodendrocyte gap junction E-faces (i.e., connexins of the underlying
oligodendrocyte cellular coupling partners) were consistently labeled
for Cx43, Cx30, or Cx30 plus Cx43 (Figs. 7-9; Table 3). In a
preliminary survey, eight oligodendrocyte cell bodies were observed
containing 12 gap junctions in their E-faces, all of which were labeled
for Cx30 and/or Cx43. Likewise, in eight myelinated processes, nine of
nine oligodendrocyte gap junction E-faces were labeled for Cx30 and/or
Cx43. In contrast, no oligodendrocyte intercelular gap junction E-faces
were labeled for Cx32 or Cx36 (Fig. 7F). This
suggested that Cx32-containing oligodendrocytes share gap junctions
with astrocytes containing both Cx30 and Cx43 but do not share gap
junctions with other oligodendrocytes or with neurons. In other
samples, as many as 27 Cx43-labeled gap junctions were found in a
single oligodendrocyte E-face, indicating that each oligodendrocyte is
coupled to many astrocyte processes.
As an independent method for identifying and quantifying the
relative numbers of oligodendrocyte-to-astrocyte,
oligodendrocyte-to-oligodendrocyte, and oligodendrocyte-to-neuron
coupling pairs, a quantitative approach was used with a limited number
of replicas having a high labeling efficiency, low background, and high
signal-to-noise ratio. To minimize investigator bias in this series of
replicas, we photographed every observed oligodendrocyte E-face having
one or more gap junctions, whether or not the gap junctions were
immunogold labeled. From those quantitative gap junction data, the
proportion of oligodendrocytes, astrocytes, and neurons as
oligodendrocyte coupling partners was determined. Plasma membrane
E-faces of 57 oligodendrocytes containing a total of 169 gap junctions
were found. Of these 169, 89% were labeled for Cx43 (Figs.
7A,C,D, 8A,B, 9A), Cx30 (Fig.
8C), or Cx43 plus Cx30 (Figs. 6B,C,
9B), thereby identifying those oligodendrocyte coupling
partners as astrocytes. In most of those examples, the identification
of Cx43-labeled oligodendrocyte coupling partners as astrocytes was
confirmed on the basis of the presence of labeled or unlabeled AQP4
arrays in their plasma membranes (Fig. 8A) or of GFAP
filaments in their cytoplasms (Fig. 8B). Included in
the 169 gap junctions are data from 19 myelinated processes in which 34 gap junctions were observed in the outermost layer of myelin. Twenty-nine (85%) of these were labeled for Cx43, Cx30, or Cx43 plus
Cx30 (Fig. 9), identifying those as oligodendrocyte myelin-to-astrocyte junctions.
An additional 8% of the 169 gap junctions in oligodendrocyte E-faces
were not labeled for either Cx43 or Cx30 but were identified as
oligodendrocyte-to-astrocyte on the basis of ultrastructural or
immunocytochemical markers. Occasionally both labeled and unlabeled gap
junctions were in close proximity (Figs. 8A,B,
9B, junctions 2, 3). Most of the remaining
coupling partners at unlabeled gap junctions were positively identified
as astrocytes, either because the unlabeled gap junction was one of two
gap junctions coupled to the same cell process, with the second gap
junction labeled for astrocytic Cx43 or Cx30 (Fig.
8A), because the plasma membrane of the coupled cell
had astrocytic AQP4 arrays (Fig. 8A), or because the
fracture plane had been diverted into the cytoplasm of the subjacent
coupled cell, where it exposed astrocytic GFAP filaments (Fig.
8A,B). Thus, of the unlabeled 11% of gap junctions
in oligodendrocyte E-faces, most (8%) were identified as heterologous
oligodendrocyte-to-astrocyte gap junctions on the basis of
immunocytochemical or ultrastructural features. Only 3% remained
unidentified, in most cases because no portion of the coupling partner
was visible in the replica. Therefore, in samples used for quantitative
analysis, >97% of oligodendrocyte gap junctions were shared with
astrocytes, and none were detected linking with other oligodendrocytes
or with neurons. Conversely, in single- and double-labeled samples,
none of the oligodendrocyte myelin coupling partners were labeled for Cx32 or Cx36, and most (98%) were labeled for Cx30 and/or Cx43, suggesting that internodal myelin shares intercellular gap junctions only with astrocytes.
With the above search strategy, a small number of
oligodendrocyte-to-oligodendrocyte gap junctions could have gone
unnoticed, particularly if unusual cell pairings had gone unexamined.
To maximize the possibility of finding homologous
oligodendrocyte-to-oligodendrocyte gap junctions, replicas were
searched for sites in which two oligodendrocyte somata were in direct
contact. Chains and clusters of oligodendrocytes occur in and near
white matter tracts, and their directly contacting somata are
frequently linked by elaborate tight junctions, often with gap
junctions observed in close association with the tight junction strands
(Massa and Mugnaini, 1982; Mugnaini, 1986). If oligodendrocyte-to-oligodendrocyte gap junctions exist, these oligodendrocyte pairs whose membranes are in molecular contact over
large areas would seem to provide the best opportunity for formation of gap junctions. However, unlike the junctional complexes in
liver and pancreas (Friend and Gilula, 1972; Wolburg and
Rohlmann, 1995), strands of oligodendrocyte-to-oligodendrocyte tight
junctions were never observed to enclose or surround gap junctions
(Fig. 7B), which would have constituted documentation of
oligodendrocyte-to-oligodendrocyte gap junctions. Instead, gap
junctions were observed outside of areas enclosed by tight junction
strands, often at distances of <1 µm (Fig. 7A,D). After
FRIL, the cellular coupling partners for these tight
junction-associated gap junctions were consistently labeled for Cx43
(Fig. 7F), as were gap junctions at slightly greater distances from the tight junctions (Fig.
7C-E). In contrast, none of the tight
junction-associated gap junctions in oligodendrocyte E-faces were
labeled for Cx32. Thus, close cellular apposition in areas linked by
tight junctions did not result in formation of homologous
oligodendrocyte-to-oligodendrocyte gap junctions.
Cx36 present on both sides of neuronal gap junctions; Cx32
not found on either side
In replicas of retina, brain, and spinal cord that were
single- or double-labeled for Cx36, 271 neuronal gap junctions were detected, 259 of which were labeled (96%; Table 3; Fig.
10). Many of these were in confirmed
neuronal E-faces coupling with cells identified as neurons because they
expressed Cx36 (Fig. 10) or had other ultrastructural markers of
neurons (data not shown). Labeled and unlabeled neuronal gap junctions
consisted of plaques of connexons (inferior olive, Fig.
10A; spinal cord, data not shown; and retina, Fig.
10B) or linear strands of connexons (retina, data not
shown). In contrast, in 229 replicas that were single-, double-, or
triple-labeled for Cx30, Cx32, and Cx43 and in which >3500 labeled gap
junctions were identified, no gap junction in neurons or their coupling
partners was labeled for any of these confirmed glial connexins.
Finally, in these labeled replicas, in >300 unlabeled replicas
examined previously (Rash et al., 1998b), as well as in all published
freeze-fracture studies, neither we nor any other group has found a
neuron whose coupling partner exhibited ultrastructural markers of
oligodendrocytes or astrocytes (see Waxman and Pappas, 1971; Massa and
Mugnaini, 1982; Landis et al., 1983; Mugnaini, 1986; Rash et al.,
1997). Thus, by FRIL and conventional freeze-fracture, we found no
evidence of neuron-to-glial gap junctions.
View larger version (178K):
[in this window]
[in a new window]
|
Figure 10.
Stereoscopic images of Cx36-labeled neuronal gap
junctions in inferior olive (A) and retina
(B). A, Neuronal gap junction
labeled for Cx36 by three 20 nm gold beads. Postsynaptic density
(arrow) is a useful marker for identifying neuronal
plasma membranes in freeze-fracture replicas (Rash et al., 1997,
2000). C, Two Cx36-labeled gap junctions in a nerve
terminal in rat retina. Postsynaptic density (arrow) is
indicated.
|
|
| |
DISCUSSION |
This study addresses several issues regarding the organization and
composition of glial and neuronal gap junctions. In contrast to
predictions of the shared-connexins/mixed-coupling hypothesis (Nadarajah et al., 1996; Alvarez-Maubecin et al., 2000), gap junctions in each of three cell types (oligodendrocytes, astrocytes, and neurons)
were shown to contain a limited subset from among the four connexins
examined. Cx32 was only in oligodendrocyte gap junctions, Cx30 and Cx43
were only in astrocytes, and Cx36 was only in neurons. Using these data
for cell-specific connexin labeling, in combination with distinctive
ultrastructural features to confirm cell identifications, we found that
(1) oligodendrocytes shared intercellular gap junctions only with
astrocytes, but not detectably with other oligodendrocytes, (2)
astrocytes shared gap junctions with astrocytes and oligodendrocytes,
but not with neurons, and (3) neurons shared gap junctions with other
neurons, but not with oligodendrocytes or with astrocytes (Fig.
11).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 11.
Diagram illustrating cellular coupling
partners and connexin constituents in their gap junctions, as
identified by FRIL. The cells linked within the glial syncytium are
indicated by light gray shading. Astrocytes
(A) share gap junctions with ependymocytes
(E), oligodendrocytes (O),
and other astrocytes. Ependymocyte-to-ependymocyte
(E) gap junctions contain Cx43 but not Cx30,
Cx32, or Cx36. Astrocyte gap junctions contain Cx43 and Cx30 but not
Cx32 or Cx36. Oligodendrocytes (O) share gap
junctions only with astrocytes; the oligodendrocyte sides of these
junctions contain Cx32 but not Cx30, Cx36, or Cx43. Neurons
(N) share gap junctions with other neurons and
not with astrocytes or oligodendrocytes. Neuronal gap junctions contain
Cx36 but not Cx30, Cx32, or Cx43.
|
|
These conclusions were based on unique investigative advantages of
FRIL. (1) Multiple immunogold beads were localized to an easily
recognized structural feature that has clearly defined borders, (2)
connexons from each of two coupled cells are always separated during
cleaving, which allowed unambiguous identification of the connexin(s)
present in each cell, uncontaminated by connexins of its cellular
coupling partner, and (3) by double or triple labeling, many gold beads
of a specific size were found on most gap junctions of one cell type
and, simultaneously, were absent from all gap junctions of other cell
types, thereby confirming the cell specificity of connexin expression
as well as providing independent tests for false positives and false
negatives. Consequently, after the cell specificities of connexin
expression were determined by FRIL, LM immunocytochemical experiments
using cell-specific double-labeling methods revealed the abundance
and subcellular distribution of gap junctions linking each cell type.
Evidence that oligodendrocytes share gap junctions only
with astrocytes
Early freeze-fracture studies found that oligodendrocytes shared
gap junctions only with astrocytes (Massa and Mugnaini, 1982; Mugnaini,
1986). However, <5% of oligodendrocyte coupling partners could be
identified by conventional freeze-fracture because replicated portions
of oligodendrocyte coupling partners were usually too small to include
defining features (Mugnaini, 1986; Rash et al., 1997). Thus, unknown
systematic artifacts of cleaving could have skewed results so that
oligodendrocyte-to-astrocyte coupling pairs were recognized but
oligodendrocyte-to-oligodendrocyte or oligodendrocyte-to-neuron coupling pairs were not detected. On the basis of the demonstration that each of four connexins was cell specific and could be used as
markers for cell identification (this report), the number of cell pairs
in which both cells were identified was increased to >97%, thereby
allowing for accurate determination of relative frequencies of cellular
coupling partners among neurons and glia. By the use of two
complementary searching methods, Cx32 was localized to the
oligodendrocytic side but not to the coupling-partner side of gap
junctions formed by oligodendrocytes, extending previous data for
cell-specific intercellular coupling of oligodendrocytes with
astrocytes (Li et al., 1997). However, the present study did not
address the composition of autologous gap junctions between successive
layers of myelin (Sandri et al., 1977; Scherer et al., 1995).
Weak electrical coupling and limited dye transfer between
oligodendrocytes have been observed in culture (Kettenmann and Ransom, 1988), in optic nerve (Butt and Ransom, 1989), and in gray matter of
early postnatal spinal cord slices, with no demonstrable dye coupling
of oligodendrocytes in white matter regions (Pastor et al., 1998).
Moreover, asymmetric or functionally rectified coupling of
oligodendrocytes with astrocytes occurs in retinal glial cells (Robinson et al., 1993) and in spinal cord gray matter (Pastor et al.,
1998). As noted by Pastor et al. (1998), these observations of dye
coupling between oligodendrocytes are consistent with ultrastructural evidence that adjacent oligodendrocytes share numerous gap junctions with astrocytes, which serve as "intermediaries" between successive oligodendrocytes, thereby permitting otherwise isolated
oligodendrocytes to participate in gap junction intercellular
communication within the broader panglial syncytium (Mugnaini, 1986;
Rash et al., 1997). The hypothesis that oligodendrocytes represent
"blind-ended side branches" of the glial syncytium, coupled to
other oligodendrocytes only via astrocyte intermediaries, arose because
of the proposed absence of oligodendrocyte-to-oligodendrocyte gap
junctions and the abundance of oligodendrocyte-to-astrocyte junctions
(Mugnaini, 1986). This report provides further support for the
hypothesis of restricted coupling of oligodendrocytes with astrocytes.
The large area of oligodendrocyte myelin around multiple axons,
together with minimal cytoplasm within the myelin sheaths, may require communication via gap junctions that connect myelin to the syncytial pool of astrocytes yet allow the requisite functional isolation of each
oligodendrocyte and its myelinating segments from other oligodendrocytes (Fig. 11).
Neuronal gap junctions and neuronal coupling partners
In classical ultrastructural studies that used strict
criteria for designating close membrane appositions as gap junctions, the number of neuron-to-neuron gap junctions was found to be low in
most areas of the CNS (Brightman and Reese, 1969; Sloper, 1972; Sotelo
and Korn, 1978), and until recently, neuron-to-glial cell gap junctions
had never been reported. Likewise, early functional studies found
evidence of only limited electrical or tracer coupling between neurons
and no evidence of direct neuron-to-glial dye transfer (for review, see
Dudek et al., 1998). Our data confirm those early views, including the
view that neuronal/glial coupling occurs by means other than gap
junctions (Parpura et al., 1994; Hassinger et al., 1995; Charles et
al., 1996). However, recent studies have concluded that
neuron-to-neuron and neuron-to-glial gap junctions are abundant,
representing 18% or even 57% of total gap junctions in cerebral
cortex and locus coeruleus (Nadarajah et al., 1996; Alvarez-Maubecin et
al., 2000). A single gold bead denoting Cx32 immunoreactivity was
reported on either or both sides of those putative neuron-to-neuron and
neuron-to-glia gap junctions, but similar low levels of Cx32 labeling
were also reported at glial gap junctions (Alvarez-Maubecin et al.,
2000). By comparison, we found previously that (1) multiple immunogold
beads (as many as 50) were at a uniform distance (<20 nm) from either
or both sides of apposed glial membranes, (2) labeled areas of
membranes were precisely parallel, with uniform separations of <3 nm,
and were of distinctly increased electron density as compared with nonlabeled membranes, and (3) the thin-section images included sufficient area to provide comparative evidence of low nonspecific background labeling (Nagy et al., 1999). The current data from FRIL
confirm and extend our descriptions from TEM immunocytochemistry.
Connexin coexistence and heterotypic coupling
On the basis of the high proportion of oligodendrocyte gap
junctions (containing Cx32) coupling with astrocytes (containing Cx30
and Cx43), most oligodendrocyte gap junctions are both heterologous (i.e., involve two different types of cells) and heterotypic (i.e., contain two or more different connexins). Supporting data from LM
immunocytochemistry include images showing that astrocytes in
vivo and in vitro express both Cx30 and Cx43 and that
Cx32 is closely associated with Cx43- and Cx30-positive puncta on
oligodendrocyte somata (Yamamoto et al., 1990a; Li et al., 1997; Nagy
et al., 1997, 1999; Ochalski et al., 1997). However, recent reports
suggest that oligodendrocytes express not only Cx32 but also Cx45
(Dermietzel et al., 1997; Kunzelmann et al., 1997) and that astrocytes
express not only Cx30 and Cx43 but also Cx45 and Cx26 (Dermietzel,
1998; Alvarez-Maubecin et al., 2000). Although we concur that
astrocytes also express Cx26, our studies of Cx45 in the CNS
remain inconclusive (J. I. Nagy and J. E. Rash, unpublished
observations). In any case, oligodendrocyte-to-astrocyte gap
junctions may contain as many as five different connexins in the
apposing plaques, with Cx32 and Cx45 in the oligodendrocyte side
coupling with Cx30, Cx43, and Cx26 and possibly Cx45 in the astrocyte
side. Precedent for three connexins in individual gap junction
hemiplaques has been shown by FRIL in heart (Severs, 1999).
The presence of Cx43 and Cx30 in the astrocyte side of
oligodendrocyte-to-astrocyte gap junctions and Cx32 in the
oligodendrocyte side is especially noteworthy because Cx43 is reported
not to form functional channels with Cx32 when expressed in
oocytes (Elfgang et al., 1995; White et al., 1995). Thus,
oligodendrocytic Cx32 may form channels with astrocytic Cx30, and
oligodendrocytic Cx45 may pair with astrocytic Cx43, both of which are
permissive combinations (White and Bruzzone, 1996). Regardless, it is
essential to determine whether oligodendrocyte or astrocyte gap
junction plaques contain Cx45 and whether, in addition to forming
channels with Cx43, oligodendrocyte Cx45 also forms functional channels
with astrocytic Cx30.
Functional considerations and unresolved issues
The functional requirement for multiple connexins at glial gap
junctions may allow for different conductance properties at different
subcellular locations, permit differential regulation of permeability
state, and allow metabolic regulation by the cells on either or both
sides of the gap junctions (White et al., 1995; Bruzzone et al., 1996;
Veenstra, 1996; White and Bruzzone, 1996). Thus, the several connexin
constituents and particular connexin pairings may impart the
distinctive coupling properties that have been observed both in
vivo and in vitro at oligodendrocyte-to-astrocyte gap
junctions (Kettenmann and Ransom, 1988; Butt and Ransom, 1989; Moreno
et al., 1991; Giaume and Venance, 1995; Sontheimer, 1995). These may
include directionality with which substances pass through gap
junctions, the low efficiency and variability observed for oligodendrocyte dye coupling, and molecular size and charge selectivity in channel permeation (Robinson et al., 1993; Veenstra, 1996; Pastor et
al., 1998). Identification of all connexins expressed at glial gap
junctions will ultimately lead to a better understanding of the
functional role of the proposed panglial syncytium in adult mammalian
CNS and, in particular, the basis for cell specificity of connexin
expression and for the exclusive sharing of intercellular oligodendrocyte gap junctions with astrocytes.
| |
FOOTNOTES |
Received Oct. 27, 2000; revised Jan. 2, 2001; accepted Jan. 3, 2001.
This work was supported by National Institutes of Health Grants
NS-31027, NS-39040, and NS-38121 to J.E.R. and MH-59995 to F.E.D. and
by grants from the Canadian Institutes of Health Research to J.I.N. We
thank G. Stelmack and D. Patel for biochemical and anatomical support
in antibody analysis and Kimberly Davidson for quantitative analysis of
labeled replicas and photographic assistance.
Higher-resolution images and additional supporting data may be viewed
at http://www.cvmbs.colostate.edu/rashlab.
Correspondence should be addressed to Dr. John E. Rash, Department of
Anatomy and Neurobiology, Colorado State University, Fort Collins, CO
80523. E-mail: jrash{at}cvmbs.colostate.edu.
| |
REFERENCES |
-
Alvarez-Maubecin V,
García-Hernández F,
Williams JT,
Van Bockstaele EJ
(2000)
Functional coupling between neurons and glia.
J Neurosci
20:4091-4098[Abstract/Free Full Text].
-
Berdan RC,
Shivers RR,
Bulloch AGM
(1987)
Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma.
Synapse
1:304-323[Web of Science][Medline].
-
Brightman MW,
Reese TS
(1969)
Junctions between intimately apposed cell membranes in the vertebrate brain.
J Cell Biol
40:648-677[Abstract/Free Full Text].
-
Bruzzone R,
White TW,
Paul DL
(1996)
Connections with connexins: the molecular basis of direct intercellular signaling.
Eur J Biochem
238:1-27[Web of Science][Medline].
-
Butt AM,
Ransom BR
(1989)
Visualization of oligodendrocytes and astrocytes in the intact rat optic nerve by intracellular injection of Lucifer yellow and horseradish peroxidase.
Glia
2:470-475[Web of Science][Medline].
-
Charles AC,
Kodali SK,
Tyndale RF
(1996)
Intercellular calcium waves in neurons.
Mol Cell Neurosci
7:337-353[Web of Science][Medline].
-
Condorelli DF,
Parenti R,
Spinella F,
Salinaro AT,
Belluardo N,
Cardile V,
Cicirata F
(1998)
Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons.
Eur J Neurosci
10:1202-1208[Web of Science][Medline].
-
Dermietzel R
(1998)
Diversification of gap junction proteins (connexins) in the central nervous system and the concept of functional compartments.
Cell Biol Int
22:719-730[Web of Science][Medline].
-
Dermietzel R,
Spray DC
(1998)
From neuro-glue ("nervenkitt") to glia: a prologue.
Glia
24:1-7[Web of Science][Medline].
-
Dermietzel R,
Farooq M,
Kessler JA,
Hertzberg EL,
Spray DC
(1997)
Oligodendrocytes express gap junction proteins connexin32 and connexin45.
Glia
20:101-114[Web of Science][Medline].
-
Dudek FE,
Yasumura T,
Rash JE
(1998)
Nonsynaptic mechanisms in seizures and epileptogenesis.
Cell Biol Int
22:793-805[Web of Science][Medline].
-
Elfgang C,
Eckert R,
Lichthenberg-Frate H,
Butterweck A
(1995)
Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.
J Cell Biol
129:805-817[Abstract/Free Full Text].
-
Friend DS,
Gilula NB
(1972)
Variations in tight and gap junctions in mammalian tissues.
J Cell Biol
53:758-776[Abstract/Free Full Text].
-
Fujimoto K
(1995)
Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.
J Cell Sci
108:3443-3449[Abstract].
-
Giaume C,
Venance L
(1995)
Gap junctions in brain glial cells and development.
Perspect Dev Neurobiol
2:335-345[Web of Science][Medline].
-
Hassinger TD,
Atkinson PB,
Strecker GJ,
Whalen LR,
Dudek FE,
Kossell AH,
Kater SB
(1995)
Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves.
J Neurobiol
28:159-170[Web of Science][Medline].
-
Hatton JD,
Ellisman MH
(1981)
The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system.
Cell Tissue Res
215:309-323[Web of Science][Medline].
-
Hudson CS,
Rash JE,
Shinowara N
(1981)
Freeze-fracture and freeze-etch methods.
In: Current trends in morphological techniques, Vol II (Johnson JE,
ed), pp 183-217. Boca Raton, FL: CRC.
-
Kettenmann H,
Ransom BR
(1988)
Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures.
Glia
1:64-73[Web of Science][Medline].
-
Kunzelmann P,
Blumcke I,
Traub O,
Dermietzel R,
Willecke K
(1997)
Coexpression of connexin45 and -32 in oligodendrocytes of rat brain.
J Neurocytol
26:17-22[Web of Science][Medline].
-
Landis DMD
(1981)
Membrane structure in mammalian astrocytes: a review of freeze-fracture studies on adult, developing, reactive and cultured astrocytes.
J Exp Biol
95:35-48[Abstract/Free Full Text].
-
Landis DMD,
Weinstein LA,
Halperin JJ
(1983)
Development of synaptic junctions in cerebellar glomeruli.
Dev Brain Res
8:231-245.
-
Li J,
Hertzberg EL,
Nagy JI
(1997)
Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain.
J Comp Neurol
379:571-591[Web of Science][Medline].
-
Massa PT,
Mugnaini E
(1982)
Cell junctions and intramembrane particles of astrocytes and oligodendrocytes: a freeze-fracture study.
Neuroscience
7:523-538[Web of Science][Medline].
-
Micevych PE,
Abelson L
(1991)
Distribution of mRNAs coding for liver and heart gap junction proteins in the rat central nervous system.
J Comp Neurol
305:96-118[Web of Science][Medline].
-
Micevych PE,
Popper P,
Hatton GI
(1996)
Connexin 32 mRNA levels in the rat supraoptic nucleus; up-regulation prior to parturition and during lactation.
Neuroendocrinology
63:39-45[Web of Science][Medline].
-
Moreno AP,
Campos de Carvalho AC,
Verselis V,
Eghbali B,
Spray DC
(1991)
Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.
Biophys J
59:920-925[Web of Science][Medline].
-
Mugnaini E
(1986)
Cell junctions of astrocytes, ependyma, and related cells in the mammalian central nervous system, with emphasis on the hypothesis of a generalized functional syncytium of supporting cells.
In: Astrocytes, Vol I (Fedoroff S,
Vernadakis A,
eds), pp 329-371. New York: Academic.
-
Nadarajah B,
Thomaidou D,
Evans WH,
Parnavelas JG
(1996)
Gap junctions in the adult cerebral cortex: regional differences in their distribution and cellular expression of connexins.
J Comp Neurol
376:326-342[Web of Science][Medline].
-
Nagy JI,
Rash JE
(2000)
Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS.
Brain Res Rev
32:29-44[Medline].
-
Nagy JI,
Ochalski PAY,
Li J,
Hertzberg EL
(1997)
Evidence for co-localization of another connexin with connexin-43 at astrocytic gap junctions in rat brain.
Neuroscience
78:533-548[Web of Science][Medline].
-
Nagy JI,
Patel D,
Ochalski PAY,
Stelmack GL
(1999)
Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance.
Neuroscience
88:447-468[Web of Science][Medline].
-
Nedergaard M
(1994)
Direct signaling from astrocytes to neurons in cultures of mammalian brain cells.
Science
263:1768-1771[Abstract/Free Full Text].
-
Nielsen S,
Nagelhus EA,
Amiry-Moghaddam M,
Bourque C,
Agre P,
Ottersen OP
(1997)
Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain.
J Neurosci
17:171-180[Abstract/Free Full Text].
-
Ochalski PAY,
Frankenstein UN,
Hertzberg EL,
Nagy JI
(1997)
Connexin43 in rat spinal cord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions.
Neuroscience
76:931-945[Web of Science][Medline].
-
Parpura V,
Basarky TA,
Liu F,
Jeftinija K,
Jeftenija S,
Haydon PG
(1994)
Glutamate-mediated astrocyte-neuron signalling.
Nature
369:744-747[Medline].
-
Pastor A,
Kremer M,
Möller T,
Kettenmann H,
Dermietzel R
(1998)
Dye coupling between spinal cord oligodendrocytes: differences in coupling efficiency between gray and white matter.
Glia
24:108-120[Web of Science][Medline].
-
Phillips TE,
Boyne AF
(1984)
Liquid nitrogen-based quick freezing: experiments with bounce-free delivery of cholinergic nerve terminals to a metal surface.
J Electron Microsc Tech
1:9-29.
-
Rash JE,
Yasumura T
(1999)
Direct immunogold labeling of connexins and aquaporin4 in freeze-fracture replicas of liver, brain and spinal cord: factors limiting quantitative analysis.
Cell Tissue Res
296:307-321[Web of Science][Medline].
-
Rash JE,
Dillman RK,
Morita M,
Whalen LR,
Guthrie PB,
Fay-Guthrie D,
Wheeler DW
(1995)
Grid-mapped freeze fracture: correlative confocal laser scanning microscopy and freeze-fracture electron microscopy of preselected cells in tissue slices.
In: Rapid freezing, freeze fracture, and deep etching (Severs NJ,
Shotton DM,
eds), pp 127-150. New York: Wiley.
-
Rash JE,
Dillman RK,
Bilhartz BL,
Duffy HS,
Whalen LR,
Yasumura T
(1996)
Mixed synapses discovered and mapped throughout mammalian spinal cord.
Proc Natl Acad Sci USA
93:4235-4239[Abstract/Free Full Text].
-
Rash JE,
Duffy HS,
Dudek FE,
Bilhartz BL,
Whalen LR,
Yasumura T
(1997)
Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system, with evidence for a "panglial syncytium" that is not coupled to neurons.
J Comp Neurol
388:265-292[Web of Science][Medline].
-
Rash JE,
Yasumura T,
Dudek FE
(1998a)
Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord.
Cell Biol Int
22:731-749[Web of Science][Medline].
-
Rash JE,
Yasumura T,
Hudson CS,
Agre P,
Nielsen S
(1998b)
Direct immunogold labeling of aquaporin-4 in "square arrays" of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord.
Proc Natl Acad Sci USA
95:11981-11986[Abstract/Free Full Text].
-
Rash JE,
Staines WA,
Yasumura T,
Patel D,
Hudson CS,
Stelmack GL,
Nagy JI
(2000)
Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin36 (Cx36) but not Cx32 or Cx43.
Proc Natl Acad Sci USA
97:7573-7578[Abstract/Free Full Text].
-
Robinson SR,
Hampson ECGM,
Munro MN,
Vaney DI
(1993)
Unidirectional coupling of gap junctions between neuroglia.
Science
262:1072-1074[Abstract/Free Full Text].
-
Sandri C,
Van Buren JM,
Akert K
(1977)
Membrane morphology of the vertebrate nervous system.
Prog Brain Res
46:1-384[Medline].
-
Scherer SS,
Deschenes SM,
Xu Y-T,
Grinspan JP,
Fischbeck KH,
Paul DL
(1995)
Connexin32 is a myelin-related protein in the PNS and CNS.
J Neurosci
15:8281-8284[Abstract].
-
Severs NJ
(1999)
Cardiovascular disease.
Novartis Found Symp
219:188-206[Web of Science][Medline].
-
Sloper JJ
(1972)
Gap junctions between dendrites in the primate cortex.
Brain Res
44:641-646[Medline].
-
Sontheimer H
(1995)
Coupling in glial cells: who is coupled, and why?
The Neuroscientist
1:188-191.
-
Sotelo C,
Korn H
(1978)
Morphological correlates of electrical and other interactions through low-resistance pathways between neurons of the vertebrate central nervous system.
Int Rev Cytol
55:67-107[Medline].
-
Veenstra RD
(1996)
Size and selectivity of gap junction channels formed from different connexins.
J Bioenerg Biomembr
28:327-337[Web of Science][Medline].
-
Waxman SG,
Black JA
(1984)
Freeze-fracture ultrastructure of the perinodal astrocyte and associated glial junctions.
Brain Res
308:77-87[Web of Science][Medline].
-
Waxman SG,
Pappas GD
(1971)
An electron microscopic study of synaptic morphology in the oculomotor nuclei of three inframammalian species.
J Comp Neurol
143:41-72[Web of Science][Medline].
-
White T,
Paul D,
Goodenough DA,
Bruzzone R
(1995)
Functional analysis of selective interactions among rodent connexins.
Mol Biol Cell
6:459-470[Abstract].
-
White TW,
Bruzzone R
(1996)
Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences.
J Bioenerg Biomembr
28:339-350[Web of Science][Medline].
-
Wolburg H,
Rohlmann A
(1995)
Structure-function relationships in gap junctions.
Int Rev Cytol
157:315-373[Web of Science][Medline].
-
Wolff JR,
Stuke K,
Missler M,
Tytko H,
Schwarz P,
Rohlmann A,
Chao TL
(1998)
Autocellular coupling by gap junctions in cultured astrocytes: a new view on cellular autoregulation during process formation.
Glia
24:121-140[Web of Science][Medline].
-
Yamamoto T,
Ochalski A,
Hertzberg EL,
Nagy JI
(1990a)
LM and EM immunolocalization of the gap junctional protein connexin43 in rat brain.
Brain Res
508:313-319[Web of Science][Medline].
-
Yamamoto T,
Ochalski A,
Hertzberg EL,
Nagy JI
(1990b)
On the organization of astrocytic gap junctions in the brain as suggested by LM and EM immunocytochemistry of connexin43 expression.
J Comp Neurol
302:853-883[Web of Science][Medline].
Copyright © 2001 Society for Neuroscience 0270-6474/01/2161983-18$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J Miguel-Hidalgo, Y Shoyama, and V Wanzo
Infusion of gliotoxins or a gap junction blocker in the prelimbic cortex increases alcohol preference in Wistar rats
J Psychopharmacol,
July 1, 2009;
23(5):
550 - 557.
[Abstract]
[PDF]
|
|
|
|
|
|
|
C. Satou, Y. Kimura, T. Kohashi, K. Horikawa, H. Takeda, Y. Oda, and S.-i. Higashijima
Functional Role of a Specialized Class of Spinal Commissural Inhibitory Neurons during Fast Escapes in Zebrafish
J. Neurosci.,
May 27, 2009;
29(21):
6780 - 6793.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Sargiannidou, N. Vavlitou, S. Aristodemou, A. Hadjisavvas, K. Kyriacou, S. S. Scherer, and K. A. Kleopa
Connexin32 Mutations Cause Loss of Function in Schwann Cells and Oligodendrocytes Leading to PNS and CNS Myelination Defects
J. Neurosci.,
April 15, 2009;
29(15):
4736 - 4749.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Orthmann-Murphy, E. Salsano, C. K. Abrams, A. Bizzi, G. Uziel, M. M. Freidin, E. Lamantea, M. Zeviani, S. S. Scherer, and D. Pareyson
Hereditary spastic paraplegia is a novel phenotype for GJA12/GJC2 mutations
Brain,
February 1, 2009;
132(2):
426 - 438.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. E. Flores, X. Li, M. V. L. Bennett, J. I. Nagy, and A. E. Pereda
Interaction between connexin35 and zonula occludens-1 and its potential role in the regulation of electrical synapses
PNAS,
August 26, 2008;
105(34):
12545 - 12550.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Orthmann-Murphy, M. Freidin, E. Fischer, S. S. Scherer, and C. K. Abrams
Two Distinct Heterotypic Channels Mediate Gap Junction Coupling between Astrocyte and Oligodendrocyte Connexins
J. Neurosci.,
December 19, 2007;
27(51):
13949 - 13957.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Hamzei-Sichani, N. Kamasawa, W. G. M. Janssen, T. Yasumura, K. G. V. Davidson, P. R. Hof, S. L. Wearne, M. G. Stewart, S. R. Young, M. A. Whittington, et al.
Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze fracture replica immunogold labeling
PNAS,
July 24, 2007;
104(30):
12548 - 12553.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Ennis and S. Datta
Electrotonic Coupling in the Nucleus SubCoeruleus. Focus on "Evidence for Electrical Coupling in the SubCoeruleus (SubC) Nucleus"
J Neurophysiol,
April 1, 2007;
97(4):
2579 - 2579.
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Menichella, M. Majdan, R. Awatramani, D. A. Goodenough, E. Sirkowski, S. S. Scherer, and D. L. Paul
Genetic and Physiological Evidence That Oligodendrocyte Gap Junctions Contribute to Spatial Buffering of Potassium Released during Neuronal Activity
J. Neurosci.,
October 25, 2006;
26(43):
10984 - 10991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. P. Schools, M. Zhou, and H. K. Kimelberg
Development of Gap Junctions in Hippocampal Astrocytes: Evidence That Whole Cell Electrophysiological Phenotype Is an Intrinsic Property of the Individual Cell
J Neurophysiol,
September 1, 2006;
96(3):
1383 - 1392.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. E. Isakson, D. N. Damon, K. H. Day, Y. Liao, and B. R. Duling
Connexin40 and connexin43 in mouse aortic endothelium: evidence for coordinated regulation
Am J Physiol Heart Circ Physiol,
March 1, 2006;
290(3):
H1199 - H1205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. McCulloch, R. Chambrey, D. Eladari, and J. Peti-Peterdi
Localization of connexin 30 in the luminal membrane of cells in the distal nephron
Am J Physiol Renal Physiol,
December 1, 2005;
289(6):
F1304 - F1312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Hewitt, R. Barrie, M. Graham, K. Bogus, J. C. Leiter, and J. S. Erlichman
Ventilatory effects of gap junction blockade in the RTN in awake rats
Am J Physiol Regulatory Integrative Comp Physiol,
December 1, 2004;
287(6):
R1407 - R1418.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. H. Stobbs, A. J. Ohran, M. B. Lassen, D. W. Allison, J. E. Brown, and S. C. Steffensen
Ethanol Suppression of Ventral Tegmental Area GABA Neuron Electrical Transmission Involves N-Methyl-D-aspartate Receptors
J. Pharmacol. Exp. Ther.,
October 1, 2004;
311(1):
282 - 289.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Venance, J. Glowinski, and C. Giaume
Electrical and chemical transmission between striatal GABAergic output neurones in rat brain slices
J. Physiol.,
August 15, 2004;
559(1):
215 - 230.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. M. Altevogt and D. L. Paul
Four Classes of Intercellular Channels between Glial Cells in the CNS
J. Neurosci.,
May 5, 2004;
24(18):
4313 - 4323.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. G. Placantonakis, A. A. Bukovsky, X.-H. Zeng, H.-P. Kiem, and J. P. Welsh
Fundamental role of inferior olive connexin 36 in muscle coherence during tremor
PNAS,
May 4, 2004;
101(18):
7164 - 7169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Meier, R. Dermietzel, K. G. V. Davidson, T. Yasumura, and J. E. Rash
Connexin32-Containing Gap Junctions in Schwann Cells at the Internodal Zone of Partial Myelin Compaction and in Schmidt-Lanterman Incisures
J. Neurosci.,
March 31, 2004;
24(13):
3186 - 3198.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Martin, T. Tawadros, L. Meylan, A. Abderrahmani, D. F. Condorelli, G. Waeber, and J.-A. Haefliger
Critical Role of the Transcriptional Repressor Neuron-restrictive Silencer Factor in the Specific Control of Connexin36 in Insulin-producing Cell Lines
J. Biol. Chem.,
December 26, 2003;
278(52):
53082 - 53089.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Gomez-Hernandez, M. de Miguel, B. Larrosa, D. Gonzalez, and L. C. Barrio
Molecular basis of calcium regulation in connexin-32 hemichannels
PNAS,
December 23, 2003;
100(26):
16030 - 16035.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N Rouach, M Segal, A Koulakoff, C Giaume, and E Avignone
Carbenoxolone blockade of neuronal network activity in culture is not mediated by an action on gap junctions
J. Physiol.,
December 15, 2003;
553(3):
729 - 745.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. A. Taylor, E. M. Simon, H. G. Marks, and S. S. Scherer
The CNS phenotype of X-linked Charcot-Marie-Tooth disease: More than a peripheral problem
Neurology,
December 9, 2003;
61(11):
1475 - 1478.
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Scheffler, T. Schmandt, W. Schroder, B. Steinfarz, L. Husseini, J. Wellmer, G. Seifert, K. Karram, H. Beck, I. Blumcke, et al.
Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures
Development,
November 15, 2003;
130(22):
5533 - 5541.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Iacobas, M. Urban-Maldonado, S. Iacobas, E. Scemes, and D. C. Spray
Array analysis of gene expression in connexin-43 null astrocytes
Physiol Genomics,
November 11, 2003;
15(3):
177 - 190.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Pereda, J. O'Brien, J. I. Nagy, F. Bukauskas, K. G. V. Davidson, N. Kamasawa, T. Yasumura, and J. E. Rash
Connexin35 Mediates Electrical Transmission at Mixed Synapses on Mauthner Cells
J. Neurosci.,
August 20, 2003;
23(20):
7489 - 7503.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Rios, M. Rubin, M. St. Martin, R. T. Downey, S. Einheber, J. Rosenbluth, S. R. Levinson, M. Bhat, and J. L. Salzer
Paranodal Interactions Regulate Expression of Sodium Channel Subtypes and Provide a Diffusion Barrier for the Node of Ranvier
J. Neurosci.,
August 6, 2003;
23(18):
7001 - 7011.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Menichella, D. A. Goodenough, E. Sirkowski, S. S. Scherer, and D. L. Paul
Connexins Are Critical for Normal Myelination in the CNS
J. Neurosci.,
July 2, 2003;
23(13):
5963 - 5973.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Odermatt, K. Wellershaus, A. Wallraff, G. Seifert, J. Degen, C. Euwens, B. Fuss, H. Bussow, K. Schilling, C. Steinhauser, et al.
Connexin 47 (Cx47)-Deficient Mice with Enhanced Green Fluorescent Protein Reporter Gene Reveal Predominant Oligodendrocytic Expression of Cx47 and Display Vacuolized Myelin in the CNS
J. Neurosci.,
June 1, 2003;
23(11):
4549 - 4559.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. S. Jahromi, K. Wentlandt, S. Piran, and P. L. Carlen
Anticonvulsant Actions of Gap Junctional Blockers in an In Vitro Seizure Model
J Neurophysiol,
October 1, 2002;
88(4):
1893 - 1902.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Duval, D. Gomes, V. Calaora, A. Calabrese, P. Meda, and R. Bruzzone
Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells
J. Cell Sci.,
August 15, 2002;
115(16):
3241 - 3251.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. M. Altevogt, K. A. Kleopa, F. R. Postma, S. S. Scherer, and D. L. Paul
Connexin29 Is Uniquely Distributed within Myelinating Glial Cells of the Central and Peripheral Nervous Systems
J. Neurosci.,
August 1, 2002;
22(15):
6458 - 6470.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. F. Bukauskas, A. Bukauskiene, V. K. Verselis, and M. V. L. Bennett
Coupling asymmetry of heterotypic connexin 45/ connexin 43-EGFP gap junctions: Properties of fast and slow gating mechanisms
PNAS,
May 14, 2002;
99(10):
7113 - 7118.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. E. Landisman, M. A. Long, M. Beierlein, M. R. Deans, D. L. Paul, and B. W. Connors
Electrical Synapses in the Thalamic Reticular Nucleus
J. Neurosci.,
February 1, 2002;
22(3):
1002 - 1009.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Contreras, H. A. Sanchez, E. A. Eugenin, D. Speidel, M. Theis, K. Willecke, F. F. Bukauskas, M. V. L. Bennett, and J. C. Saez
Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture
PNAS,
December 21, 2001;
(2001)
12589799.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Contreras, H. A. Sanchez, E. A. Eugenin, D. Speidel, M. Theis, K. Willecke, F. F. Bukauskas, M. V. L. Bennett, and J. C. Saez
Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture
PNAS,
January 8, 2002;
99(1):
495 - 500.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
