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The Journal of Neuroscience, June 1, 2000, 20(11):4091-4098
Functional Coupling between Neurons and Glia
Veronica
Alvarez-Maubecin1,
Fernando
García-Hernández2,
John T.
Williams1, and
Elisabeth J.
Van
Bockstaele2
1 Vollum Institute for Advanced Biomedical Research,
Oregon Health Science University, Portland, Oregon 97201, and
2 Department of Pathology, Anatomy, and Cell Biology,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
Neuronal-glial interactions play an important role in information
processing in the CNS. Previous studies have indicated that electrotonic coupling between locus ceruleus (LC) neurons is
involved in synchronizing the spontaneous activity. The results of the present study extend the functional electrotonic coupling to
interactions between neurons and glia. Spontaneous oscillations in the
membrane potential were observed in a subset of glia. These
oscillations were synchronous with the firing of neurons, insensitive
to transmitter receptor antagonists and disrupted by carbenoxolone, a
gap junction blocker. Hyperpolarization of neurons with [Met]
5enkephalin blocked the oscillations in glia.
Selective depolarization of glia with a glutamate transporter substrate
(L- -aminoadipic acid) increased the neuronal firing
rate, suggesting that changes in the membrane potential of glia can
modulate neuronal excitability through heterocellular coupling.
Dye-coupling experiments further confirmed that small molecules could
be transferred through gap junctions between these distinct cell types.
No dye transfer was observed between neurons and oligodendrocytes or
between astrocytes and oligodendrocytes, suggesting that the junctional
communication was specific for astrocytes and neurons. Finally,
immunoelectron microscopy studies established that connexins, the
proteins that form gap junctions, were present on portions of the
plasmalemma, bridging the cytoplasm of neurons and glia in LC. This
heterocellular coupling extends the mechanisms by which glia
participate in the network properties of the LC in which the degree of
coupling is thought to influence cognitive performance.
Key words:
electrotonic coupling; gap junction; neuronal-glial
interactions; membrane potential oscillations; noradrenergic neurons; connexins
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INTRODUCTION |
Neuronal-glial interactions
typically require chemical signaling through the extracellular space
(Parpura et al., 1994 ; Coles and Abbott, 1996; Theodosis and MacVicar,
1996; Bezzi et al., 1998). For example, exogenous application or
synaptically released neurotransmitters activate receptors on glia to
affect their membrane potential, evoke second messenger signals, and
induce calcium waves (Dani et al., 1992; Duffy and MacVicar, 1995;
Verkhratsky and Kettenmann, 1996; Chen et al., 1997; Pasti et al.,
1997). In turn, glia remove neurotransmitters from the extracellular space and maintain proper ionic balance (Jendelova and Sykova, 1991;
Bergles et al., 1999). Moreover, glutamate released from glia has been
shown to modulate evoked and spontaneous synaptic transmission in
neurons (Araque et al., 1998). Together, these bidirectional
interactions mediate neuronal-glial communication and indicate that
glia play an active role in influencing neuronal activity.
Direct coupling through gap junctions has been suggested previously to
mediate communication between neurons and glia. In cultures from
embryonic brain, stimulation of calcium waves in astrocytes was shown
to induce propagated waves in neurons (Nedergaard, 1994).
Heterocellular electrotonic coupling was the proposed mechanism (but
see Parpura et al., 1994); however, electrical coupling was not tested
and dye transfer was not observed. A recent report has now demonstrated
that, in embryonic neuronal cultures, neurons were coupled to
astrocytes but that coupling declined progressively with maturation and
was rarely detectable in cultures from postnatal neurons (Froes et al.,
1999).
The purpose of the present study was to test the hypothesis that
heterocellular coupling exists in brain slices taken from postnatal
animals. The combination of immunoelectron microscopy and
electrophysiology was used to evaluate neuronal-glial interactions and
its functional consequences in the noradrenergic brain nucleus locus
ceruleus (LC). Electrotonic coupling between neurons in the LC is known
to regulate neuronal activity (Christie et al., 1989; Christie and
Jelinek, 1993). In the present report, neurons and glia were found to
be coupled as demonstrated by the presence of gap junction proteins and
both electrical and dye transfer between the two cell types. Moreover,
activation of glutamate transporters selectively depolarized glia and
indirectly increased neuronal excitability.
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MATERIALS AND METHODS |
Electrophysiology
Horizontal brainstem slices (250 µm) containing the LC were
prepared from 4- to 10-d-old Sprague Dawley rats (Charles River, Wilmington, MA) as described previously (Eghbali et al., 1990). Whole-cell recordings were made using Nomarski optics and infrared visualization that allowed morphological discrimination of different cell types (Fig. 1A).
Slices were perfused with artificial CSF (ACSF) containing (in
mM):126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and 11.1 glucose, equilibrated with 95%O2-5%CO2 at 34°C. Tetrodotoxin (1 µM) and 500 µM
BaCl2 were added to ACSF during the recordings.
Local application of glutamate was conducted by pressure ejection of a
saline solution (140 mM NaCl and 10 mM HEPES) containing glutamate (1 mM). The pipettes used had a resistance of 1-2
M , the duration of the pulses was 100 msec, and the pressure applied
was 4 psi. In some experiments, sodium was replaced with lithium in the
ACSF in the same concentration, 126 mM.
1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-silfonamide (NBQX) and -amino-4-carboxy--methyl-phenylacetic acid
(MCPG) were obtained from Tocris Cookson (Ballwin, MO),
picrotoxin, L-aminoadipic acid
(L-AA), and carbenoxolone were from Sigma (St.
Louis, MO), and
5-methyl-10,11-dihydro-5H-debenzo[a,d]cyclohepten-5,10-imine hydrogen
meleate (MK-801) and
1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) were from Research Biochemicals (Natick, MA).
Pipettes (2-3 M) were filled with internal solution containing (in
mM): 115 MES(2-[morpholino]ethane-sulfonic
acid) potassium salt, 20 KCl, 1.5 MgCl2, 1 BAPTA,
5 HEPES, 4 Mg-ATP, and 0.4 Na-GTP, pH 7.3. Recordings were made
with an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) in
current-clamp mode, and data were acquired at 100 Hz with Chart
software, version 3.5 (MacLab System; ADInstruments, Castle Hill,
Australia).
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Figure 1.
Rhythmic oscillations in membrane potential of
neurons and glia in the LC. A, High-power view of the LC
in a brainstem slice from 1-week-old rat using infrared illumination.
The differences in size and morphology between LC neurons and glia
allowed selective recordings from each cell type. Scale bar, 20 µm.
B, Left, Representative recordings from a
neuron and a glial cell made in the presence of tetrodotoxin (1 µM) and BaCl2 (500 µM).
Right, Power spectrum analyses of the membrane
potential. For the glia, the peak was 0.58 Hz; for the neuron, the peak
was 0.51 Hz.
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Experiments using acutely isolated LC neurons were done as described
previously (Ingram et al., 1997). Drug administration was performed
with a fast flow exchange consisting of a series of tubes driven by a
piezoelectric device.
Immunocytochemistry
Rat pups were anesthetized with sodium pentobarbital and
transcardially perfused through the ascending aorta with 10 ml of 1000 U/ml heparinized saline, 50 ml of 3.75% acrolein in 2%
paraformaldehyde, and 100 ml of 2% paraformaldehyde in 0.1 M phosphate buffer (PB) at a pH of 7.4. A pre-embedding
double-labeling method was used (Chan et al., 1990). Tissue sections
were post-fixed for 1 hr in 2% osmium tetroxide in 0.1 M
PB, washed for 10 min in PB, dehydrated through a graded series of
ethanols and propylene oxide, and then transferred to a 1:1 mixture of
propylene oxide and Epon 812 overnight. Sections were then incubated in
Epon 812 for 2 hr and flat embedded in Epon between plastic sheets for
12-18 hr at 60°C. Small pieces of tissue were sampled from the LC
and re-embedded in Beem capsules. The avidin-biotin peroxidase
technique was visualized using 3'-3' diaminobenzidine as a chromogen
and was used to identify connexin (Cx) proteins, tyrosine
hydroxylase (TH), or glial fibrillary acidic protein (GFAP).
Immunogold-silver labeling (Chan et al., 1990) was combined with
immunoperoxidase to simultaneously localize two antigens. Gold-silver
labeling of the Cx proteins was conducted to confirm the peroxidase
localization because peroxidase markers are known to have a tendency to
diffuse within the tissue. It should be noted, however, that minimum
peroxidase diffusion was observed here. Sections of horizontal brain
processed after primary antibody omission served as controls. Alternate
horizontal sections were processed for all experiments in which the
markers were reversed to confirm immunolabeling results with each
technical approach.
Specificity of antisera. Two antibodies directed against
Cx32 [a mouse monoclonal 2C2 from Zymed (San Francisco, CA) and
MAB3069 from Chemicon, (Temecula, CA)] were tested for specificity in rat liver and positively identified gap junctions as described by
others (Paul, 1986; Dermietzel et al., 1990). Both antibodies are
raised against the cytoplasmic loop of rodent Cx32, and the monoclonal
2C2 antibody labeling was shown to have a comparable distribution with
that seen with other noncommercially available antibodies (Li et al.,
1997). The antibodies directed against Cx26 and Cx43 were also obtained
from Zymed. A rabbit polyclonal antibody raised against TH (Protos
Biotech, New York, NY) or a mouse monoclonal antibody directed against
TH (Incstar, Stillwater, MN) was used to recognize noradrenergic
neurons of the LC. Specificity of these antibodies has been confirmed
(Van Bockstaele et al., 1995). A rabbit polyclonal antibody directed
against GFAP was obtained from Incstar.
Quantification of immunoreactivity. Sections for
ultrastructural analysis included the LC cell body region and LC
dendritic zone. Quantitative evaluation of immunoreactive elements was
applied only to the outer 1-3 µm of the Epon-tissue interface at
which penetration of antisera is maximal. Examination of serial
sections was used to determine specificity of immunogold labeling. The classification of identified cellular elements in the electron micrographs was as described by Peters et al. (1991). All profiles identified within randomly selected electron micrographs were tabulated
with respect to immunoreactive versus nonimmunoreactive cellular
profiles. These profiles were tallied for every 3500 µm2 of sampled area, and the data were
expressed as percent values of relative frequency occurrence of a
specific type of profile per unit area.
Dye-coupling experiments
A single glial cell was patched and filled with neurobiotin
(0.2%; Vector Laboratories, Burlingame, CA) per slice. To reduce the
background staining produced by neurobiotin being ejected from the
pipette tip while approaching the cell under positive pressure,
unconjugated streptavidin (0.1%; Jackson ImmunoResearch, West Grove,
PA) was perfused in the bath. Immediately after the seal between the
pipette and the cell was made, normal ACSF perfusion was restored.
Recordings were made for 10-20 min, and then the slice was immediately
fixed in Lana's solution (paraformaldehyde 4% and picric acid 14% in
PBS, pH 6.9) overnight at 4°C. Tissue was washed in PBS,
incubated for 24-48 hr with 1:3000 dilution Cy5-conjugated
streptavidin (Jackson ImmunoResearch) in PBS-0.3%Triton X-100, at
4°C. Slices were thoroughly washed in PBS and mounted. For the
triple-labeling experiments, the fixative solution was washed from the
slices, and they were incubated with a mixture of antibodies against
neuronal- and astrocytic-specific markers and Cy5-streptavidin for 48 hr at 4°C. The cocktail contained mouse anti-S100 (1:2000; Sigma),
rabbit anti-TH (1:2000; Protos Biotech), and Cy-5-conjugated
streptavidin 1:3000 in 0.1 M PB, pH 7.4, with 0.1% BSA and
0.3% Triton X-100. Slices were washed four times in 0.1 M
PBS for 30 min and then incubated for 24 hr at 4°C with the secondary
antibodies containing FITC-conjugated donkey anti-mouse (1:200) and
Rhodamine Red-X-conjugated donkey anti-rabbit (1:200) (both from
Jackson ImmunoResearch) in 0.1 M PBS. Slices were washed in
0.1 M PBS four times and mounted with Slow Fade
(Molecular Probes, Eugene, OR). A series of images was taken with a
confocal microscope [z-series; Bio-Rad, Hercules, CA and Nikon, Tokyo,
Japan; and a Bio-Rad MCR10242P and an Olympus Optical (Tokyo, Japan)
1X70], and they were combined to reconstruct stained cells through the
extent of the slice.
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RESULTS |
Neurons and glia are electrically coupled in the LC
It is known from previous studies that LC neurons are
spontaneously active and fire action potentials in synchrony (Christie et al., 1989). This latter property was attributed to electrotonic coupling between LC neurons, which has been demonstrated by current and
dye transfer between LC neurons (Christie et al., 1989; Christie and
Jelinek, 1993). As a consequence of both electrotonic coupling and
spontaneous activity, synchronous firing and rhythmic oscillations in
the membrane potential were always present in slices from neonatal animals. The subthreshold rhythmic oscillations in the membrane potential have been used as a measure of electrotonic coupling in the
LC (Christie et al., 1989; Ishimatsu and Williams, 1996).
Recordings from glial cells in brainstem slices from 1-week-old rats
showed that the membrane potential of 27 of 52 (52%) cells exhibited
rhythmic changes in membrane potential that were similar in frequency
to neuronal oscillations (mean frequency in glia, 0.51 ± 0.04 Hz,
n = 27; in neurons, 0.47 ± 0.05 Hz,
n = 22) (Fig. 1). The mean amplitude of the
oscillations observed in glia was 0.36 ± 0.03 mV
(n = 27), 4-5% of the amplitude of the oscillations
in neurons (7.8 ± 0.6 mV, n = 22). No
oscillations were observed in small non-noradrenergic neurons found in
close proximity to the cell body region of the LC. The oscillations in
membrane potential recorded in glia suggested that these cells were
electrically coupled to neurons and that they might participate in the
LC network.
Glial cells were initially distinguished from neurons by their relative
smaller size (~10 µm diameter compared with 30-40 µm diameter
for neurons). Once the patches were made, depolarizing current pulses
were applied to confirm the absence of action potentials. In most
cells, the membrane potential responded linearly to the current
injection (n = 14 of 20) with some cells displaying
inward rectification when depolarized beyond 0 mV. The average input resistance (Rin) was 46.7 ± 5.9 M, and  82.8 ± 1.2 mV was the average resting membrane
potential. Increasing the extracellular potassium concentration from
2.5 to 15 mM shifted the membrane potential to
46 ± 1 mV, without significant change in
Rin (43.5 ± 12 M). This
depolarization (35 mV) is in reasonable agreement with the value
predicted by the Nernst equation for a potassium-selective dependence
of the membrane potential (~ 47 mV). A second group of glial cells
displayed complex I-V curves and higher
Rin (147 ± 28 M). A previous
study on astrocytes from hippocampal slices described three similar
subpopulations distinguished by electrophysiological properties,
localization, and coupling properties (D'Ambrosio et al., 1998).
Although the group of glial cells with lower resistance (presumed
astrocytes) seemed to have oscillations in the membrane potential,
correlation between electrophysiological properties and the occurrence
of oscillations was equivocal.
Calculations based on the Rin of
astrocytes and neurons (~200 M) established
that 15 pA would produce a change in membrane potential that was
similar to the oscillations in each cell type. This current was used to
estimate the number of gap junction channels between neurons and glia.
The driving force for the current transfer was calculated as the
amplitude of the afterhyperpolarization after the action potential
(~ 20 mV) because this slow potential change is less sensitive to
resistance capacitance filtering imposed by the neuronal-glial
network. The conductance required to cause the observed change in
membrane potential during a single oscillation was calculated to be
~750 pS. By assuming the unitary conductance of gap junctional
channels to be 120-150 pS (Eghbali et al., 1990), it was predicted
that five to six gap junction channels would be present on each
electrotonically coupled neuron and glia.
The rhythmic oscillations in glia persisted after perfusion of
glutamate receptor antagonists (10 µM NBQX, 10 µM MK-801, and 1 mM MCPG), the GABA receptor
antagonist bicuculline (10 µM), and tetrodotoxin (1 µM; n = 3; data not shown). Thus, glial
oscillations were independent of synaptic activity as similarly
described for LC neurons (Christie et al., 1989; Ishimatsu and
Williams, 1996). Changes in extracellular
K+ concentration are known to occur during
neuronal activity and could change the membrane potential of glia
(Orkand et al., 1966). By increasing the
K+ concentration in the extracellular
solution from 2.5 to 10 mM, only a slight
reduction (10-15%) of the amplitude of the afterhyperpolarization was
observed in neurons, without any clear effect on the firing frequency.
In addition, neither the amplitude nor the frequency of the
oscillations in glia were significantly affected by increasing the
extracellular concentration, suggesting that oscillations in glia are
not attributable to fluctuations of extracellular potassium
concentration (control amplitude of 1.25 mV, control frequency of 0.4 Hz; in 10 mM KCl, amplitude of 1.76 mV, frequency of 0.3 Hz).
Two additional experiments were conducted to identify the source of
oscillations in glia. First, [Met]
5enkephalin (ME) (10 µM), an
opioid agonist that hyperpolarizes neurons, abolished oscillations in
both neurons and glia (Fig. 2A). There was no clear
effect of ME on glial membrane potential. This result suggested that
the spontaneous activity of neurons was required to observe
oscillations in the membrane potential of glia. Second, disruption of
the coupling with carbenoxolone, a glycyrrhetinic acid analog that
blocks gap junctional communication (Ishimatsu and Williams, 1996;
Vaney et al., 1998), abolished the oscillations in both glia and
neurons (Fig. 2B). Carbenoxolone did not block
spontaneous neuronal action potentials but did desynchronize firing of
LC (see also Travagli et al., 1995). This result further suggested that it is not the spontaneous firing but the synchronous firing of LC neurons that drives the rhythmic oscillations in glia.
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Figure 2.
Oscillations in glia depend on synchronized
neuronal activity. A, Left, ME, a
µ-opioid receptor agonist, hyperpolarizes the membrane potential of
LC neurons and reversibly inhibits the subthreshold oscillations
(n = 10). Right, ME reversibly
abolishes membrane potential oscillations in glia
(n = 6). Note that ME did not induce a change in
the resting membrane potential of glia. B, Carbenoxolone
(100 µM) disrupted oscillations in both glial and
neuronal membrane potential. The effect of carbenoxolone reversed
within 30 min of washout. All recordings were made in the presence of
tetrodotoxin (1 µM) and BaCl2 (500 µM).
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If the oscillations recorded from glia were a consequence of
electrotonic coupling to neurons, then the oscillations should be
synchronous in both cells. In paired recordings from neurons and glia,
both the depolarizing and the hyperpolarizing phase of the oscillations
in glia strongly correlated with the changes in neuronal membrane
potential (cross-correlation, mean peak of 0.30 ± 0.05, mean
phase shift of 11 ± 0.4 msec, n = 6) (Fig.
3A). Moreover, oscillations in
glia were observed in 6 of 10 neuronal-glial pairs and always in
preparations in which oscillations in neurons were also recorded. As it
has been shown previously (Christie et al., 1989), the subthreshold
oscillations in LC neurons were also synchronized throughout the
nucleus (cross correlation, mean peak of 0.86 ± 0.03, mean phase
shift of 7.3 ± 1.9 msec, n = 11) (Fig.
3B). Thus, paired recordings showed that the membrane
potential of both LC neurons and LC glia oscillate rhythmically and
synchronously.
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Figure 3.
Synchronized oscillations in neurons and glia.
A, Left, A paired whole-cell recording
shows that glial oscillations are synchronous with neuronal activity.
Right, Cross-correlogram: peak of 0.29, phase shift of
10 msec. B, Left, A paired whole-cell
recording from two LC neurons. Right, Cross-correlogram:
peak of 0.94, phase shift of 10 msec.
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Selective depolarization of glia increases
neuronal excitability
To determine whether heterocellular coupling could have a
functional effect on neurons, the effect of a glial-selective agent on
neuronal excitability was examined. A glutamate analog that is a
substrate for glutamate transporters, L-AA, has been used as a glial toxin (McBean, 1994). Superfusion with L-AA (1 mM) depolarized glial cells by 7-10 mV (Fig.
4A). This
depolarization persisted in the presence of the glutamate receptor
antagonists NBQX and MK-801 but was completely blocked by substitution
of sodium by lithium in the extracellular solution (Fig.
4A). Local application of glutamate, by pressure
ejection, also depolarized glial cells by a mechanism that was
insensitive to glutamate receptor antagonists and was also blocked by
replacing sodium with lithium in the extracellular solution. The sodium
dependence and the resistance to glutamate receptor antagonists
suggested that the depolarization of glia caused by
L-AA was mediated by activation of electrogenic glutamate transporters.
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Figure 4.
Selective depolarization of glia increases
neuronal firing. A, L-AA depolarized the
membrane potential of glia through activation of glutamate
transporters. Top trace, Recording from a glial cell in
the presence of ionotropic glutamate receptor antagonists NBQX (5 µM) and MK-801 (5 µM). Bottom
trace, In same cell, substitution of sodium for lithium in the
extracellular solution completely blocks glial depolarization.
B, The effect of L-AA on neuronal firing
frequency. Right, Recordings from an LC neuron before
(Control, top trace) and during
(bottom trace) L-AA application in the
presence of NBQX (5 µM) and MK-801 (5 µM).
Left, Average change induced by L-AA on the
firing frequency of LC neurons, expressed as percentage of the control
firing rate. The mean firing frequency in control was 0.59 ± 0.21 Hz. Statistical differences were determined by the nonparametric
Wilcoxon signed rank test. *p < 0.05 indicates
significant difference.
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When L-AA was tested in neurons, it caused a
depolarization, an effect that was sensitive to glutamate receptor
antagonists. Recordings made from acutely isolated LC neurons showed
that L-AA induced an inward current that reversed polarity
at 0 mV and was completely blocked by the ionotropic glutamate receptor
antagonist NBQX (10 µM; n = 3; data not
shown). When neurons in brain slices were tested with
L-AA in the presence of glutamate receptor
antagonists, it depolarized glial cells and increased the firing
frequency by 60-90% (Fig. 4B). This increase of
neuronal excitability was reversible upon washing
L-AA, and it was present even in very high
concentrations of receptor antagonists (Fig. 4B).
Thus, activation of glutamate transporters selectively depolarized
glial cells, and that depolarization increased the firing rate of
neurons by a mechanism that was insensitive to glutamate receptor
antagonists. This experiment suggests that the electrotonic coupling
between neurons and glia can result in modulation of neuronal activity.
Locus ceruleus neurons and glia form a coupled network
Dye coupling
In experiments in which a single glial cell per slice was filled
with neurobiotin, fluorescent localization of neurobiotin showed that,
in 7 of 13 (54%) slices, neurons were also stained (Fig.
5A,B).
When a single neuron was filled, dye transfer to glia and/or other
neurons was also observed, although less frequently (19%,
n = 16). These results demonstrate that small molecules can be transferred between astrocytes and neurons through intercellular channels.
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Figure 5.
Dye coupling from glia to neurons.
A, B, Representative pictures of dye
transfer from glia to neurons in brain slices in which a single glial
cell was filled with neurobiotin. LC neurons (large
arrows) appeared stained together with several dozen smaller
cells with astrocytic morphology (small arrows).
C, Dye transfer between astrocytes was observed in all
the slices in which an astrocyte was filled (n = 13). D, Oligodendrocytes were stained in 10 of 23 slices
(43%) in which a single glial cell was filled. In all these cases,
only the filled cell was stained. Scale bar, 20 µm.
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In all the slices in which a single astrocyte was filled (13 of 23),
several dozen astrocytes were also stained (Fig. 5C), making
it impossible to distinguish the cell that was filled originally. In
the other 10 slices, only a single cell was found and the morphology corresponded to oligodendroglia (Fig. 5D). This observation
is in agreement with previous reports indicating low electrical
coupling in oligodendrocytes (Kettenmann and Ransom, 1988). In
addition, the proportion of glial cells that showed no dye coupling is
similar to glial cells that did not have oscillations in the membrane potential (48%, see above). This suggests that a correlation between the degree of coupling and the morphology may exist.
Triple-labeling experiments confirmed the identity of the different
cell types participating in the network. Slices were stained with
antibodies against an astrocytic marker, S-100 (Fig.
6, green), and TH to identify
LC neurons (blue). The extent of diffusion of neurobiotin
between cells is shown in red. In the experiment shown in
Figure 6, a recording from a single glial cell was made. That cell had
a linear I-V (Rin of 16.7 M; resting membrane potential of 80 mV). When the
extracellular potassium concentration was increased to 15 mM, the slope of the I-V curve was
not significantly changed (13.8 M), and the membrane potential was
depolarized to 46 mV. Neurobiotin was found in tens of small cells
(Fig. 6C), which colocalized with the green
immunofluorescence of the astrocytic marker S-100 (Fig.
6B, yellow). Two bigger cells were also
stained with neurobiotin and colocalized TH (Fig. 6B,
purple). Thus, in this slice, neurobiotin diffused between
many astrocytes as well as neurons.
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Figure 6.
Cellular identity of dye-coupled cells in the LC.
A, Triple staining with FITC-conjugated antibodies
against the astrocytic marker S-100 (green),
Rhodamine Red-X-conjugated antibodies against the neuronal marker
tyrosine hydroxylase (blue), and Cy-5-conjugated
streptavidin (red) to reveal neurobiotin localization.
B, Higher magnification of the area surrounding the
neurobiotin injection site. Multiple yellow cells
indicate the colocalization of the astrocytic marker and neurobiotin.
Two purple cells indicate colocalization of TH and
neurobiotin. C shows the same field of view as in
B with only the neurobiotin stain. Scale bar, 40 µm.
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Presence of connexin immunoreactivity
Immunoelectron microscopy studies were conducted to further
evaluate the anatomical substrate of gap junctional-mediated coupling between neurons and glia. Brainstem sections containing the LC of 1- to
3-week-old rats were processed for peroxidase labeling of connexins and
combined with immunogold-silver identification of TH. Peroxidase
labeling of Cx26 and Cx32 were identified at pairs of TH-immunoreactive
dendritic processes (Fig. 7A).
Similarly, when gold-silver labeling was used to identify Cx32 and
immunoperoxidase was used to localize TH, connexin immunoreactivity was
found along the plasmalemma of apposed TH dendrites (Fig.
7B,C). Cx32 and Cx26 were localized
to neuronal membranes in 19 and 12% of the profiles examined,
respectively (Fig. 7G), and in both cases, the
immunolabeling was consistently restricted to the dendrites. Thus, this
structural data establishes the physical basis underlying neuronal
coupling that has been demonstrated previously by electrophysiological studies in the LC.
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Figure 7.
Electron micrographs showing the
ultrastructural localization of Cx32 using peroxidase or gold-silver
labeling in the LC. A, A pair of adjacent dendrites
exhibiting gold-silver labeling for TH (arrowheads) also
exhibits peroxidase labeling for Cx32 (straight open
arrows) along portions of their apposed plasma membranes.
B, Gold-silver labeling for Cx32 (straight open
arrows) also reveals the presence of Cx32 on both sides of
paired apposed peroxidase-labeled TH dendrites (TH-d)
whose membranes (small filled arrows) tend to approach
at the point at which the connexin proteins are localized.
C, Gold-silver labeling for Cx32 (straight open
arrow) can be detected along the plasma membrane of apposed
peroxidase TH dendrites. One of the TH dendrites is also apposed to a
third TH dendrite (small filled arrows), which lacks Cx
immunolabeling. D, Two gold-silver
(arrowheads) TH-positive dendrites are separated from
one another by a glial process (asterisks) that exhibits
peroxidase labeling for Cx32 (straight open
ar-row). E, F, Serial
sections in which gold-silver labeling for Cx32 (straight open
arrows) was identified on the cytoplasmic surface of a
peroxidase-labeled TH dendrite and in an apposed glial process
(asterisks) that separates two TH-positive dendrites
from one another. Scale bars: A, 0.86 µm;
B, E, F, 0.2 µm;
C, 0.37 µm; D, 0.62 µm.
G, Pie charts illustrating the distribution of Cx32- and
Cx26-immunoreactive profiles pooled from peroxidase and gold-silver
stained tissue. For Cx32, 203 immunolabeled profiles were examined
across three ultrathin sections from three animals. For Cx26, 89 profiles were analyzed across three ultrathin sections from two
animals. The Cx immunoreactivity grouped in Others
corresponds to Cx32 localized to apposed membranes of glia and axon
terminals and Cx32 in association with myelinated axons. The 8% of
immunoreactivity for Cx26 was identified between glia and axons
terminals.
|
|
Cx43 immunostaining was most commonly identified in glial processes;
however, immunostaining for Cx32 and Cx26 was also frequently observed
(39% for Cx32 and 58% for Cx26) (Fig. 7G). Glial cells were identified by many of the features described for
protoplasmic, fibrous astrocytes, and oligodendrocytes, including
location in the gray matter and irregular perimeters conforming to the
shape of neighboring neuronal processes. Sometimes the astrocytic
processes contained bundles of microfilaments. These characteristics
readily distinguished glial processes from neuronal processes
exhibiting TH immunoreactivity.
Interestingly, Cx32 and Cx26 were immunocytochemically localized to the
plasma membranes of pairs of glial and dendritic processes (32 and 21%
for Cx32 and Cx26, respectively) (Fig. 7G). Peroxidase labeling of Cx32 was associated with extensions of protoplasmic astrocytes directly apposed to TH-labeled dendritic processes (Fig.
7D). Gold-silver labeling for Cx32 established the
localization of gap junction proteins to the cytoplasmic surface of
TH-labeled dendrites, as well as to plasmalemmal surfaces of astrocytic
processes. Figure 7, E and F, shows consecutive
serial sections in which Cx32 was localized with gold-silver labeling
to portions of apposed glial and neuronal plasma membranes. A previous
report described a similar neuronal-glial interaction in neocortex
(Nadarajah et al., 1996). Thus, the gap junction proteins were found at
neuronal-glial pairs, further indicating the existence of a widespread
network in the LC.
Finally, the immunostaining studies also revealed a peculiar
distribution of astrocytic processes (Fig.
8). Using immunostaining of GFAP, a
protein present in astrocytic processes, it was possible to observe
that astrocytic processes extended along a substantial portion of the
perimeter of TH-labeled dendrites. These processes were not separated
by any other cellular elements as determined from electron microscopic
analysis (data not shown). A similar distribution was found when
fluorescent immunostaining of another astrocytic marker, S-100, was
combined with fluorescent identification of TH (Fig.
8B). The striking enrichment and distribution of
astrocytes in this area constitutes another piece of evidence that
supports the idea that astrocytes might play a distinctive role in the LC.
View larger version (131K):
[in this window]
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|
Figure 8.
Distribution of astrocytic markers in the LC.
A, Immunogold-silver labeling (black) for
GFAP and peroxidase labeling for TH (brown) in the LC in
a horizontal brain section. Note how GFAP-positive processes envelop
TH-labeled cell bodies (black arrows). Scale bar,
160 µm. B, Immunofluorescent staining of S-100
(green, left) and TH
(red, center) in the LC.
Right, Superimposed image of both wavelengths showing
the absence of colocalization and the enrichment of astrocytic
processes surrounding LC neurons. Scale bar, 20 µm.
|
|
| |
DISCUSSION |
This study demonstrates gap junctional coupling between neurons
and glia in a brainstem noradrenergic nucleus. The identification of
connexin proteins and functional gap junctions by electrical and
chemical coupling demonstrates heterocellular coupling in the brain.
This novel mechanism for neuronal-glial communication adds a potential
regulatory role for glia in information processing.
On one hand, heterocellular coupling constitutes a pathway whereby
electrical signals can be transferred from neurons to glia. Although it
is unlikely that the small oscillations in membrane potential of glia
have an impact on glial cell physiology, the oscillations constitute a
convenient measure of the electrical coupling to neurons. The
electrical coupling between glia and neurons does, however, affect
neuronal excitability. Activation of glutamate transporters selectively
depolarized glial cells and increased the firing rate of LC neurons.
Synaptically released glutamate could therefore have two different
actions on LC neurons: one direct, through activation of postsynaptic
receptors, and another indirect heterocellular action mediated by the
activation of glutamate transporters on glia.
It is also probable that heterocellular coupling exerts an opposite
influence on neuronal excitability. Given the negative membrane
potential of glia (80 mV), heterocellular coupling would constitute
an electrical shunt and a steady hyperpolarizing current leak into
neurons. The tonic hyperpolarization would modulate the firing
frequency and facilitate the synchronization of the spontaneous
activity. According to previous reports, electrical coupling is
stronger in early stages of development (Peinado et al., 1993; Chang et
al., 1999). In fact, during the first 3 weeks of life, the rate of
spontaneous firing of LC neurons increased gradually from 0.3 to 3 Hz.
This fact could be a consequence of decreased coupling to glia and then
decreased shunt in adult animals.
Chemical signals are also transferred through gap junctions. It has
been well established that inositol triphosphate
(IP3)-dependent calcium waves propagate
via gap junctions between glial cells (Charles et al., 1993). Potential
chemical signaling through heterocellular coupling may involve
molecules such as IP3 and calcium, which could
effectively regulate neuronal excitability. Thus, receptor-mediated signaling in glia may have an important influence on neuronal activity.
Finally, transfer of small molecules via gap junctions could also play
an important role in support of the growth and development of the huge
axonal arbors that stem from the highly divergent LC neurons. More
recently, a correlation between the release of ATP into the
extracellular space and the expression of connexins in cell lines has
been demonstrated (Cotrina et al., 1998). These results suggested that
ATP was released through connexin hemichannels. Propagating waves of
calcium release in these cultures resulted from activation of purine
receptors by ATP rather than through electrotonic coupling (Cotrina et
al., 1998). This may be another mechanism by which astrocytic connexins
modulate activity of LC neurons because both ATP (Harms et al., 1992;
Shen and North, 1993) and adenosine (Shefner and Chiu, 1986; Regenold
and Illes, 1990) receptors are functional in LC neurons.
Synchronous activity in the locus ceruleus is one mechanism by which
the release of noradrenaline in the widespread projection areas can be
augmented. A recent report showed that changes in LC activity pattern
correlated with fluctuation in the performance of a visual
discrimination task in waking monkeys (Usher et al., 1999). Periods of
high performance in the behavioral task were associated with a
reduction in the tonic discharge and an increase of the phasic
discharge of LC neurons. Interestingly, the phasic activity was found
to be synchronous in recordings made from pairs of cells. A
computational model showed that modulation of the degree of
electrotonic coupling of LC neurons was a potentially important factor
in predicting the response of the animals. The present work expands the
cellular network present in the LC to include heterocellular
interactions with glia. Although the present work demonstrated
heterocellular coupling in neonatal animals, qualitatively similar
results were obtained in preliminary studies in slices from older
animals. Thus, the regulation of activity through electrotonic coupling
may extend into adulthood.
| |
FOOTNOTES |
Received Dec. 28, 1999; revised March 9, 2000; accepted March 17, 2000.
This work was supported by National Science Foundation Grant 9810524 and National Institutes of Health/National Institute on Drug Abuse
Grant DA04523. We thank Spencer Watts and Dwight Bergles for the
invaluable technical advice, and Olivier Manzoni, Linda Musil,
Peregrine Osborne, Cecilia Lo, and Bruce Nicholson for the helpful
comments on this manuscript.
Correspondence should be addressed to Elisabeth J. Van Bockstaele,
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson
University, 1020 Locust Street, Room 520, Philadelphia, PA 19107. E-mail: elisabeth.vanbockstaele{at}mail.tju.edu.
| |
REFERENCES |
-
Araque A,
Sanzgiri RP,
Parpura V,
Haydon PG
(1998)
Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons.
J Neurosci
18:6822-6829[Abstract/Free Full Text].
-
Bergles DE,
Diamond JS,
Jahr CE
(1999)
Clearance of glutamate inside the synapse and beyond.
Curr Opin Neurobiol
9:293-298[Web of Science][Medline].
-
Bezzi P,
Carmignoto G,
Pasti L,
Vesce S,
Rossi D,
Rizzini BL,
Pozzan T,
Volterra A
(1998)
Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
Nature
391:281-285[Medline].
-
Chan J,
Aoki C,
Pickel VM
(1990)
Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding.
J Neurosci Methods
33:113-127[Web of Science][Medline].
-
Chang Q,
Gonzalez M,
Pinter MJ,
Balice-Gordon RJ
(1999)
Gap junctional coupling and patterns of connexin expression among neonatal rat lumbar spinal motor neurons.
J Neurosci
19:10813-10828[Abstract/Free Full Text].
-
Charles AC,
Dirksen ER,
Merrill JE,
Sanderson MJ
(1993)
Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin.
Glia
7:134-145[Web of Science][Medline].
-
Chen J,
Backus KH,
Deitmer JW
(1997)
Intracellular calcium transients and potassium current oscillations evoked by glutamate in cultured rat astrocytes.
J Neurosci
17:7278-7287[Abstract/Free Full Text].
-
Christie MJ,
Jelinek HF
(1993)
Dye-coupling among neurons of the rat locus coeruleus during postnatal development.
Neuroscience
56:129-137[Web of Science][Medline].
-
Christie MJ,
Williams JT,
North RA
(1989)
Electrical coupling synchronizes subthreshold activity in locus coeruleus neurons in vitro from neonatal rats.
J Neurosci
9:3584-3589[Abstract].
-
Coles JA,
Abbott NJ
(1996)
Signalling from neurones to glial cells in invertebrates.
Trends Neurosci
19:358-362[Web of Science][Medline].
-
Cotrina ML,
Lin JHC,
Alves-Rodrigues A,
Liu S,
Li J,
Azmi-Ghadimi H,
Kang J,
Naus CCG,
Nedergaard M
(1998)
Connexins regulate calcium signaling by controlling ATP release.
Proc Natl Acad Sci USA
95:15735-15740[Abstract/Free Full Text].
-
D'Ambrosio R,
Wenzel J,
Schwartzkroin PA,
McKhann GM,
Janigro II D
(1998)
Functional specialization and topographic segregation of hippocampal astrocytes.
J Neurosci
18:4425-4438[Abstract/Free Full Text].
-
Dani JW,
Chernjavsky A,
Smith SJ
(1992)
Neuronal activity triggers calcium waves in hippocampal astrocyte networks.
Neuron
8:429-440[Web of Science][Medline].
-
Dermietzel R,
Hwang TK,
Spray DS
(1990)
The gap junction family: structure, function and chemistry.
Anat Embryol
182:517-528[Medline].
-
Duffy S,
MacVicar BA
(1995)
Adrenergic calcium signaling in astrocyte networks within the hippocampal slice.
J Neurosci
15:5535-5550[Abstract].
-
Eghbali B,
Kessler JA,
Spray DC
(1990)
Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.
Proc Natl Acad Sci USA
87:1328-1331[Abstract/Free Full Text].
-
Froes MM,
Correia AH,
Garcia-Abreu J,
Spray DC,
Campos de Carvalho AC,
Neto MV
(1999)
Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures.
Proc Natl Acad Sci USA
96:7541-7546[Abstract/Free Full Text].
-
Harms L,
Finta EP,
Tschopl M,
Illes P
(1992)
Depolarization of rat locus coeruleus neurons by adenosine 5'-triphosphate.
Neuroscience
48:941-952[Web of Science][Medline].
-
Ingram S,
Wilding TJ,
McCleskey EW,
Williams JT
(1997)
Efficacy and kinetics of opioid action on acutely dissociated neurons.
Mol Pharmacol
52:136-143[Abstract/Free Full Text].
-
Ishimatsu M,
Williams JT
(1996)
Synchronous activity in locus coeruleus results from dendritic interactions in pericoerulear regions.
J Neurosci
16:5196-5204[Abstract/Free Full Text].
-
Jendelova P,
Sykova E
(1991)
Role of glia in K+ and pH homeostasis in the neonatal rat spinal cord.
Glia
4:56-63[Web of Science][Medline].
-
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].
-
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].
-
McBean GJ
(1994)
Inhibition of the glutamate transporter and glial enzymes in rat striatum by the gliotoxin, alpha aminoadipate.
Br J Pharmacol
113:536-540[Web of Science][Medline].
-
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].
-
Nedergaard M
(1994)
Direct signaling from astrocytes to neurons in cultures of mammalian brain cells.
Science
263:1768-1771[Abstract/Free Full Text].
-
Orkand RK,
Nicholls JG,
Kuffler SW
(1966)
Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia.
J Neurophysiol
29:788-806[Free Full Text].
-
Parpura V,
Basarsky TA,
Liu F,
Jeftinija K,
Jeftinija S,
Haydon PG
(1994)
Glutamate-mediated astrocyte-neuron signalling.
Nature
369:744-747[Medline].
-
Pasti L,
Volterra A,
Pozzan T,
Carmignoto G
(1997)
Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ.
J Neurosci
17:7817-7830[Abstract/Free Full Text].
-
Paul DL
(1986)
Molecular cloning of cDNA for rat liver gap junction protein.
J Cell Biol
103:123-134[Abstract/Free Full Text].
-
Peinado A,
Yuste R,
Katz LC
(1993)
Extensive dye coupling between rat neocortical neurons during the period of circuit formation.
Neuron
10:103-114[Web of Science][Medline].
-
Peters A,
Josephson K,
Vincent SL
(1991)
Effects of aging on the neuroglial cells and pericytes within area 17 of the rhesus monkey cerebral cortex.
Anat Rec
229:384-398[Medline].
-
Regenold JT,
Illes P
(1990)
Inhibitory adenosine A-1-receptors on rat locus coeruleus neurones: an intracellular electrophysiological study.
Naunyn Schmiedebergs Arch Pharmacol
341:225-231[Web of Science][Medline].
-
Shefner SA,
Chiu TH
(1986)
Adenosine inhibits locus coeruleus neurons: an intracellular study in a rat brain slice preparation.
Brain Res
366:364-368[Web of Science][Medline].
-
Shen KZ,
North RA
(1993)
Excitation of rat locus coeruleus neurons by adenosine 5'-triphosphate: ionic mechanism and receptor characterization.
J Neurosci
13:894-899[Abstract].
-
Theodosis DT,
MacVicar B
(1996)
Neurone-glia interactions in the hypothalamus and pituitary.
Trends Neurosci
19:363-367[Web of Science][Medline].
-
Travagli RA,
Dunwiddie TV,
Williams JT
(1995)
Opioid inhibition in locus coeruleus.
J Neurophysiol
74:519-528[Web of Science].
-
Usher M,
Cohen JD,
Servan-Schreiber D,
Rajkowski J,
Aston-Jones G
(1999)
The role of locus coeruleus in the regulation of cognitive performance.
Science
283:549-554[Abstract/Free Full Text].
-
Van Bockstaele EJ,
Branchereau P,
Pickel VM
(1995)
Morphologically heterogeneous met-enkephalin terminals form synapses with tyrosine hydroxylase-containing dendrites in the rat nucleus locus coeruleus.
J Comp Neurol
363:423-438[Web of Science][Medline].
-
Vaney DI,
Nelson JC,
Pow DV
(1998)
Neurotransmitter coupling through gap junctions in the retina.
J Neurosci
18:10594-10602[Abstract/Free Full Text].
-
Verkhratsky A,
Kettenmann H
(1996)
Calcium signalling in glial cells.
Trends Neurosci
19:346-352[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20114091-08$05.00/0
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|
|
|
|
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|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
|
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[Full Text]
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
T. Fauquier, N. C. Guerineau, R. A. McKinney, K. Bauer, and P. Mollard
Folliculostellate cell network: A route for long-distance communication in the anterior pituitary
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July 17, 2001;
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|
|
|
|
TOP
ABSTRACT
INTRODUCTION
