J Neurophysiol 98: 2878 –2886, 2007.
First published September 12, 2007; doi:10.1152/jn.00362.2007.
Gap Junctions Are Required for NMDA Receptor–Dependent Cell Death
in Developing Neurons
Juan Carlos de Rivero Vaccari,1 Roderick A. Corriveau,3 and Andrei B. Belousov2,4
1
Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center; 2Department of Cell and Molecular
Biology, Tulane University, New Orleans, Louisiana; 3Coriell Institute for Medical Research, Camden, New Jersey; and 4Department of
Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas
Submitted 30 March 2007; accepted in final form 11 September 2007
INTRODUCTION
In the developing nervous system, coupling of neurons by
gap junctions is abundant and plays an important role in
neurogenesis (Becker and Mobbs 1999), neuronal differentiation (Bani-Yaghoub et al. 1999), neuroblast migration (Lo
Turco and Kriegstein 1991), synaptogenesis, and neural circuit
formation (Kandler and Katz 1995; Peinado et al. 1993). Gap
junction coupling decreases during later stages of development
as chemical synapses mature (Arumugam et al. 2005; Connors
et al. 1983; Kandler and Katz 1995) and the decrease is
mediated by N-methyl-D-aspartate (NMDA) receptor activity
through the Ca2⫹/cyclic adenosine monophosphate (cAMP)
response element binding protein-dependent signaling pathway
(Arumugam et al. 2005). In the mature nervous system, gap
junction coupling increases during traumatic injury and ischemia (Chang et al. 2000; de Pina-Benabou et al. 2005; Frantseva
et al. 2002a; Scemes and Spray 1998). The death of neurons
also may occur during development, trauma, and ischemia
Address for reprint requests and other correspondence: A. B. Belousov,
Department of Molecular and Integrative Physiology, University of Kansas
Medical Center, 2146 W. 39th Avenue, M/S 3051, Kansas City, KS 66160
(E-mail: [email protected]).
2878
(Buss and Oppenheim 2004; Choi 1996; Cole and Perez-Polo
2004), i.e., when gap junction coupling is high. However,
whether gap junctions contribute to death of neural cells
(neurons and glia) or are protective is still controversial (Perez
Velazquez et al. 2003).
The NMDA receptor, a glutamate-gated ion channel, plays a
critical role in neuronal development and has been extensively
studied for its role in adult models of learning and memory
(Contestabile 2000; Goodman and Shatz 1993; Nakazawa et al.
2004; Scheetz and Constantine-Paton 1994). In vivo and
in vitro studies indicate that normal endogenous levels of
NMDA receptor activity are required for neuronal survival,
and too much or too little NMDA receptor function causes
neuronal cell death (Adams et al. 2004; Arundine and Tymianski 2004; de Rivero Vaccari et al. 2006; Hardingham and
Bading 2003; Waxman and Lynch 2005; Yoon et al. 2003). For
example, the absence or blockade of NMDA receptors in vivo
increases neuronal apoptosis in the developing brain during the
peak of naturally occurring cell death and synaptogenesis
(Adams et al. 2004; de Rivero Vaccari et al. 2006; Ikonomidou
et al. 1999). On the other hand, in traumatic injuries and
ischemia excessive amounts of glutamate are released, thus
causing NMDA receptor hyperactivity and also neuronal cell
death due to excitotoxicity (Arundine and Tymianski 2004;
Biegon et al. 2004; Giza et al. 2006). Although some data
regarding signaling mechanisms that underlie NMDA receptor– dependent regulation of cell death are available (Adams
et al. 2004; Arundine and Tymianski 2004; de Rivero Vaccari
et al. 2006; Waxman and Lynch 2005; Yoon et al. 2003), it is
unknown whether gap junctions contribute to these mechanisms. Here we investigate the role of gap junctions in neuronal cell death that is caused by both NMDA receptor hypofunction and NMDA receptor hyperfunction in developing
hypothalamic neurons. Our studies demonstrate that gap junctions, particularly those containing neuronal connexin 36
(Cx36), contribute to neuronal cell death caused by either
increasing or decreasing NMDA receptor function.
METHODS
Animal care
The use of animal subjects in these experiments was approved by
the Tulane University Animal Care and Use Committee. All experiThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0022-3077/07 $8.00 Copyright © 2007 The American Physiological Society
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
de Rivero Vaccari JC, Corriveau RA, Belousov AB. Gap junctions
are required for NMDA receptor– dependent cell death in developing
neurons. J Neurophysiol 98: 2878 –2886, 2007. First published September 12, 2007; doi:10.1152/jn.00362.2007. A number of studies
have indicated an important role for N-methyl-D-aspartate (NMDA)
receptors in cell survival versus cell death decisions during neuronal
development, trauma, and ischemia. Coupling of neurons by electrical
synapses (gap junctions) is high or increases in neuronal networks
during all three of these conditions. However, whether neuronal gap
junctions contribute to NMDA receptor–regulated cell death is not
known. Here we address the role of neuronal gap junction coupling in
NMDA receptor–regulated cell death in developing neurons. We
report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In
contrast, increasing or decreasing NMDA receptor function when gap
junction coupling is low has no or greatly reduced impact on cell
survival. Pharmacological inactivation of gap junctions or knockout of
neuronal connexin 36 prevents the cell death caused by NMDA
receptor hypofunction or hyperfunction. The results indicate the
critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons.
Based on these data, we propose the novel hypothesis that NMDA
receptors and gap junctions work in concert to regulate neuronal
survival.
ROLE OF GAP JUNCTIONS IN NMDA RECEPTOR-DEPENDENT CELL DEATH
2879
Sigma–Aldrich). Some cultures were not treated and served as control. In all cultures, the culture medium was changed twice a week.
Animals
Neuronal survival analysis
Sprague–Dawley rats and Cx36 knockout and wild-type mice were
used in these experiments. The Cx36 knockout was originally created
by Dr. David Paul (Harvard Medical School) and was donated to us
by Dr. Marla Feller (University of California, San Diego). In the Cx36
knockout, the Cx36 coding sequence is replaced by a LacZ-IRESPLAP reporter cassette (Deans et al. 2001); activation of the Cx36
promoter results in expression of the cytoplasmic protein ␤-galactosidase and not Cx36. Cx36 knockout and wild-type mice were kept as
homozygous colonies. For mouse genotyping, tail samples were
collected from each animal. Samples were lysed in buffer overnight at
55°C, then 100 ␮l of HPLC 䡠 H2O was added, and the material was
boiled for 10 min. The extracted DNA was precipitated in 1 ml of
isopropanol and resuspended in 150 ␮l of HPLC 䡠 H2O overnight at
65°C. PCR analysis was done using a set of primers for the Cx36
wild-type band (5⬘-AGC GGA GGG AGC AAA CGA GAAG-3⬘ and
5⬘-CTG CCG AAA TTG GGA ACA CTG AC-3⬘; 533 bp product)
and the Cx36 knockout band (5⬘-TCC GGC CGC TTG GGT
GGAG-3⬘ and 5⬘-CAG GTA GCC GGA TCA AGC GTA TGC-3⬘;
360 bp product) to determine the genotype.
Neuronal survival was analyzed at the end of the treatment period
(e.g., on DIV17 for DIV14 –17 treatment) using a toxicity assay as
described (Belousov et al. 2001). In brief, cells on coverslips were
washed in buffer and stained for 30 min with 1 ␮M calcein AM
(Sigma–Aldrich); this staining labels only live cells. Coverslips were
then washed and mounted on a glass slide. Photographs from 60
randomly chosen fields were taken in each coverslip using an Olympus BX51 microscope (⫻20 magnification), 490 nm (FITC) filter, and
ORCA-285 digital camera (50 ms, camera exposure time). Because
bleaching of calcein fluorescence was relatively fast (⬃15 s), this
helped to prevent the double photographing of the same fields. The
number of live neurons was counted in the obtained photographs
using OpenLab software (Improvision). Neurons had the characteristic rounded cell body, small size (⬃20 ␮m), and multiple tiny
processes and were easily distinguished from astrocytes that did not
have a clear-cut cell body and processes, were spread, and had a
bigger size (40 –100 ␮m). Each test was done blindly on at least four
coverslips from four independent cultures.
Confocal microscopy and dye transfer
Culture preparation
Neuronal cultures were prepared as described (Belousov et al.
2001) from embryonic day 18 –19 medial hypothalamus (obtained
from rats or mice). Pregnant animals were anesthetized with Nembutal
before embryos were removed. After disaggregation using papain,
cells were resuspended in standard tissue culture medium, triturated to
form a single cell suspension, and plated on glass coverslips
(⬃250,000 of cells per coverslip). To ensure that the same number of
cells was plated on each coverslip, the following procedures were
done. First, the sample of cell suspension medium was diluted two
times with Trypan Blue dye-exclusion medium. After such a treatment, live cells stay uncolored while the dead cells appear blue under
the microscope. Second, the number of live cells was counted in five
large squares in a hemacytometer (Hausser Scientific), in four corner
squares and in the middle square. The number of cells was estimated
using the following formula: the number of cells per coverslip
(⬃250,000) ⫽ (total number of cells in 5 squares, ⬃1,250)/(chamber
depth, 0.1 mm)/(the total surface area counted, 5 mm2) ⫻ (dilution
factor, 2) ⫻ (volume of the cell suspension plated on a coverslip, 5 ⫻
10⫺2 ml). Third, if the number of cells in suspension was too high or
too low, the volume of the plated cells was adjusted. Cultures were
raised in glutamate- and glutamine-free minimal essential medium
(Invitrogen) with supplements (Belousov et al. 2001). Cytosine ␤-Darabinofuranoside selectively inhibits DNA synthesis, but does not
affect RNA synthesis (Parson et al. 1995). It does not kill dividing
cells, but suppresses their proliferation. To suppress proliferation of
glial cells, cytosine ␤-D-arabinofuranoside (1 ␮M) was added to the
culture medium on day in vitro 1 (DIV1; in cultures treated/tested on
DIV1– 4 and in the corresponding controls) or DIV2 (in all other
cultures).
DIV15 untreated (control) cultures were loaded for 30 min at 37°C
with calcein AM (125 ␮M) in a standard perfusion solution containing: 137 mM NaCl, 25 mM glucose, 10 mM HEPES, 5 mM KCl, 1
Pharmacological treatments
Pharmacological treatments were done in sister cultures for 3, 13,
17, or 27 days as indicated herein. The following pharmacological
agents were added to the culture medium (either alone or in combinations): dizocilpine maleate (MK-801, 20 ␮M), D,L-2-amino-5-phosphonovalerate (AP5, 100 ␮M), 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 10 ␮M), glutamate (100 ␮M), NMDA (100 ␮M), bicuculline
(Bic, 50 ␮M), picrotoxin (PiTX, 500 ␮M), carbenoxolone (CBX, 25
␮M), and 18␣-glycyrrhetinic acid (18-GA, 20 ␮M) (all obtained from
J Neurophysiol • VOL
FIG. 1. Change in the number of neurons in rat hypothalamic cultures over
time. A and B: live neurons that are fluorescently stained with calcein AM
(green color, A) and the corresponding bright field image (B) are shown
[images from day in vitro (DIV) 14 control cultures are demonstrated].
C: graph shows the number of live neurons per microscope field (p.m.f.) in
control cultures over time. Data are presented as mean ⫾ SE (n ⫽ 6 or more
for each test). Statistical significance was estimated using one-way ANOVA
with post hoc Tukey–Kramer.
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
ments were conducted in accordance with guidelines issued by the US
National Institutes of Health.
2880
DE RIVERO VACCARI, CORRIVEAU, AND BELOUSOV
mM MgCl2, 3 mM CaCl2, 1 ␮M glycine (aerated with 95% O2-5%
CO2 at pH 7.4). The coverslip was then washed in a perfusion solution
and held in a laminar-style chamber (Warner Instrument, Hamden,
CT) that allows for rapid (5–10 s) and complete change in the
medium. Cells were continuously perfused, using a flow pipe perfusion system (Belousov et al. 2001), initially with the perfusion
solution and then for 25 min with solutions containing NMDA (100
Statistical analysis
Data were analyzed using ANOVA with post hoc Tukey test or
paired Student’s t-test and InStat software. The data are presented as
means ⫾ SE. The numerical values are expressed according to this
formula: percentage of live neurons per microscope field (p.m.f.) ⫽
(mean number of live neurons in 60 microscope fields in a coverslip) ⫻ 100/(mean number of live neurons in 60 fields in a coverslip
with the control culture).
RESULTS
Neuronal death caused by NMDA receptor hypofunction in
developing neurons
We studied the role of gap junctions in neuronal cell death
caused by NMDA receptor hypofunction in rat hypothalamic
neuronal cultures. To visualize living neurons we used calcein
AM, the fluorescent dye that labels live cells (Fig. 1, A and B).
In untreated (control) cultures, the number of live neurons
p.m.f. decreased between DIV1 and DIV14, but it did not
significantly change between DIV14 and DIV31 (Fig. 1C).
Previous experiments showed that, in these cultures, gap junction coupling is high on DIV15–19 and is absent or minimal on
DIV1– 4 and DIV28 –31 as estimated by dye coupling (neurobiotin) and expression of the neuronal connexin, Cx36 (Arumugam et al. 2005). A 3-day treatment with the NMDA
receptor antagonists AP5 or MK-801 was performed in cultures on DIV1– 4, DIV14 –17, and DIV28 –31. NMDA receptor
blockade reduced the number of live neurons p.m.f. on
DIV14 –17 (Fig. 2B), but did not affect neuronal survival on
FIG. 2. A 3-day blockade of gap junctions in rat hypothalamic cultures
prevents neuronal cell death caused by inactivation of N-methyl-D-aspartate
(NMDA) receptors. A and C: treating cultures with D,L-2-amino-5-phosphonovalerate (AP5) or dizocilpine maleate (MK-801) on DIV1– 4 (A) or
DIV28 –31 (C) does not induce neuronal cell death. B: treating cultures with
AP5, MK-801, or AP5/6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on
DIV14 –17 induces cell death, which is prevented by coincubation with the gap
junction blockers carbenoxolone (CBX) and 18␣-glycyrrhetinic acid (18-GA).
CNQX added on DIV14 –17 does not affect neuronal survival. CBX and
18-GA applied alone do not affect neuronal survival under all conditions
(A–C). Here and in Figs. 3–5: the data are presented as means ⫾ SE (n ⫽ 4 or
more for each test); statistical significance (one-way ANOVA with post hoc
Tukey–Kramer) is shown relative to the control (*) or the primary treatment
(#) [e.g., AP5, MK-801, and AP5/CNQX in Figs. 2, 3, 5; or glutamate, NMDA,
and bicuculline/picrotoxin (Bic/PiTX) in Figs. 4 and 5].
J Neurophysiol • VOL
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
␮M) or AP5 (100 ␮M) (all solutions were aerated and applied at a
constant perfusion rate of 1.5 ml/min at room temperature, 20 –22°C).
Laser scanning confocal microscopy was performed using a Nikon C1
Plus microscope, Nikon ⫻60 oil objective, and Nikon EZ-C1 software. Calcein fluorescence was visualized by excitation at 488 nm
with the argon laser and emission fluorescence was collected at
505–525 nm. Images were taken before and 25 min after the beginning of drug application. Intensity of calcein fluorescence was expressed as absolute fluorescence intensity in arbitrary units (0 – 4,096);
in different neurons the fluorescence intensity varied between 950 and
3,000. In each group, the analysis was done in six coverslips (in one
microscope field per coverslip) obtained from three independent
culture preparations. Each microscope field contained 17–22 neurons
that were easily distinguished from astrocytes as described earlier.
The fluorescence intensity was measured in a neuronal cell body. The
concentration of calcein AM chosen for these experiments (125 ␮M)
was in the same range as in the previous similar study in neuronal
cultures (Thompson et al. 2006); in that study, 25 min also was more
than sufficient to detect the ischemia-induced hemichannel opening.
ROLE OF GAP JUNCTIONS IN NMDA RECEPTOR-DEPENDENT CELL DEATH
2881
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
either DIV1– 4 or DIV28 –31 (Fig. 2, A and C). The cell death
caused by either AP5 or MK-801 was prevented by the gap
junction blockers CBX and 18-GA (Fig. 2B). Adding AP5 plus
CNQX (a non-NMDA glutamate receptor antagonist) to cultures yielded results similar to those obtained with AP5 or
MK-801 (Fig. 2B). Treatment of neurons with CNQX, CBX,
and 18-GA (alone or in combinations) did not affect neuronal
survival (Fig. 2B). A more prolonged blockade of NMDA
receptors on DIV4 –17 or DIV14 –31 did not affect neuronal
survival (Fig. 3, A and B), although survival increased during
NMDA receptor blockade on DIV4 –31 (Fig. 3C). Further,
during these prolonged treatments, the survival of neurons was
not affected by CBX under any of the conditions tested (Fig. 3,
A–C). The results indicate that gap junctions are not necessary
for neuronal survival, that NMDA receptor hypofunction induces cell death during a specific critical period (in this case
DIV14 –17), and that this increase in cell death requires gap
junctions.
Neuronal death caused by NMDA receptor hyperfunction in
developing neurons
We also studied the role of gap junctions in neuronal cell
death induced by glutamate and NMDA receptor hyperfunction. Rat hypothalamic neuronal cultures were treated with
glutamate to activate all glutamate receptors, NMDA to activate NMDA receptors only, or a combination of Bic and PiTX
to block ␥-aminobutyric acid type A (GABAA) receptors, thus
to increase glutamate-mediated synaptic transmission and the
activity of synaptic glutamate receptors (Hardingham and Bading 2002). None of these manipulations affected survival of the
neurons when the drugs were present in the cultures on
DIV1– 4 (Fig. 4A). Administration of these drugs on DIV14 –
17, however, induced cell death that was prevented by the gap
junction blockers CBX and 18-GA (Fig. 4B). When added to
cultures on DIV28 –31, glutamate induced cell death, but the
effects of NMDA and Bic/PiTX were either undetectable
(Bic/PiTX) or statistically insignificant (NMDA) (Fig. 4C). In
contrast to the results obtained for DIV14 –17, inactivation of
gap junctions with CBX at DIV28 –31 did not inhibit the
excitotoxic effect of glutamate. As expected, the effects of
glutamate, NMDA, and Bic/PiTX were also prevented by
NMDA receptor antagonists alone (AP5 or MK-801) or in
combination with a non-NMDA receptor antagonist (CNQX)
(Fig. 4D). In addition, neither CBX nor 18-GA affected cell
survival when added to cultures alone (Fig. 4, A–C). The
results confirm that NMDA receptor hyperfunction is the
critical element in glutamate-dependent excitotoxicity in developing neurons and indicate that NMDA receptor–mediated
cell death requires gap junctions.
Role of Cx36 in NMDA receptor– dependent cell death
Cx36 is a gap junction protein that is neuron-specific and is
essential for functional gap junction coupling between hypothalamic neurons (Belluardo et al. 2000; Long et al. 2005; Rash
et al. 2000). We used hypothalamic cultures obtained from
wild-type and Cx36 knockout mice to test whether elimination
of the neuronal connexin affects cell death caused by decreased
or increased NMDA receptor function. As in rat hypothalamic
J Neurophysiol • VOL
FIG. 3. Prolonged inactivation of NMDA receptors in rat hypothalamic cultures does not affect or increases neuronal survival. A–C: inactivation of NMDA
receptors on DIV4 –17 (A), DIV14 –31 (B), and DIV4 –31 (C). CBX does not
affect neuronal survival under all conditions. Sample size: n ⱖ 4 for each test.
cultures (Figs. 2 and 4), DIV14 –17 treatment of wild-type
mouse hypothalamic cultures with AP5, NMDA, or Bic/PiTX
all caused neuronal cell death that was prevented by the
blockade of gap junctions with CBX (Fig. 5). Moreover,
98 • NOVEMBER 2007 •
www.jn.org
2882
DE RIVERO VACCARI, CORRIVEAU, AND BELOUSOV
neither NMDA receptor hypofunction nor NMDA receptor
hyperfunction affected neuronal survival in cultures prepared
using Cx36 knockout mice (Fig. 5). The results confirm a
critical role for gap junctions, in particular Cx36-containing, in
NMDA receptor– dependent regulation of survival in developing neurons.
Are hemichannels involved?
DISCUSSION
Role of gap junctions in NMDA receptor– dependent
cell death
NMDA receptors play a critical role in regulating neuronal
survival and either too much or too little of NMDA receptor
activity leads to increased neuronal cell death: the neurodegeneration caused by NMDA receptor excitotoxicity is well documented (Arundine and Tymianski 2004; Waxman and Lynch
2005); the absence or inactivation of NMDA receptors also
increases neuronal cell death in the developing brain (Adams
et al. 2004; de Rivero Vaccari et al. 2006; Yoon et al. 2003).
Here we address the role of gap junctions in the NMDA
receptor–regulated cell death caused by either increased or
decreased NMDA receptor function in developing hypothalamic neurons. We demonstrate that both inactivation and
hyperactivation of NMDA receptors induce neuronal cell death
in hypothalamic cultures and this occurs at the time when
neuronal gap junction coupling is high (i.e., DIV14 –17; Arumugam et al. 2005). The neuronal cell death caused by inactivation of NMDA receptors is completely prevented by pharmacological blockade of gap junctions. Additionally, such
NMDA receptor hypofunction-mediated cell death does not
FIG. 4. A 3-day blockade of gap junctions in rat hypothalamic cultures
prevents neurodegeneration caused by hyperactivation of NMDA receptors.
A: treating cultures with glutamate, NMDA, or Bic/PiTX on DIV1– 4 does not
induce neuronal cell death. B: treating cultures with these agents on DIV14 –17
induces neurodegeneration, which is prevented by the gap junction blockers
CBX and 18-GA. C: only application of glutamate induces neurodegeneration
on DIV28 –31 and this is not affected by CBX. CBX or 18-GA applied alone
does not affect neuronal survival under all conditions (A–C). D: on DIV14 –17,
NMDA receptor antagonists prevent neuronal cell death caused by glutamate,
NMDA, and Bic/PiTX. Sample size: n ⱖ 4 for each test.
J Neurophysiol • VOL
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
It has been reported that open hemichannels may play a role
in mediating neuronal cell death. For example, ischemic-like
conditions open pannexin hemichannels, and it has been suggested that this contributes to cell death during ischemia
(Thompson et al. 2006). Moreover, connexin 32 hemichannels
open in a Ca2⫹-dependent manner that results in release of
ATP (De Vuyst et al. 2006). Although there is no evidence that
mammalian Cx36 forms hemichannels, fish connexin 35 (Cx35),
which is a member of the Cx35/Cx36 subgroup of connexins,
does form hemichannels (Al-Ubaidi et al. 2000; Valiunas et al.
2004). We tested the possibility that hemichannels open during
activation or inactivation of NMDA receptors in developing
neurons, thus contributing to NMDA receptor– dependent regulation of neuronal cell death during development. We loaded DIV15
untreated (control) neurons with calcein AM, a fluorescent dye
that is known to pass through hemichannels (Thompson et al.
2006), to measure calcein efflux from neurons. No change in
calcein fluorescence was detected in neurons after administration
of NMDA (Fig. 6, A, B, and E) or AP5 (Fig. 6, C–E). This
suggests that neuronal hemichannels are unlikely to contribute to
NMDA receptor–regulated cell death in developing hypothalamic
neurons.
ROLE OF GAP JUNCTIONS IN NMDA RECEPTOR-DEPENDENT CELL DEATH
2883
occur at the time when gap junction coupling is absent or
minimal (on DIV1– 4 or DIV28 –31; Arumugam et al. 2005).
Thus the results suggest that gap junctions play a major role in
neuronal cell death caused by inactivation of NMDA receptors.
Similarly, the neuronal death caused on DIV14 –17 by hyperactivation of NMDA receptors is prevented by the pharmacological blockade of gap junctions. The NMDA receptor
hyperactivity-induced neurotoxicity is not observed in cultures
when gap junction coupling is absent or minimal (i.e., DIV1– 4
and DIV28 –31; Arumugam et al. 2005). Interestingly, similar
effects are observed when synaptic glutamate receptors are
hyperactivated during neuronal disinhibition with the GABAA
receptor antagonists: such a treatment causes neurodegeneration in cultures only at the time when gap junction coupling is
high (DIV14 –17) and the neurodegeneration is prevented by
pharmacological gap junction coupling blockade. As for the
neurodegenerative effect caused by the application of glutamate, it progresses over the age of cultures: it is not observed
in young cultures (DIV1– 4) and is detected after DIV14 –17
and DIV28 –31 treatments, although the effect is lower when
gap junction coupling is also low (DIV28 –31). Thus our
results suggest that gap junctions play an important role in
neuronal cell death caused by hyperactivation of synaptic
glutamate receptors, and NMDA receptors specifically.
Cx36 is the major connexin in neurons, including the hypothalamic neurons (Belluardo et al. 2000; Long et al. 2005; Rash
et al. 2000; Sohl et al. 1998). The expression of Cx36 in the
CNS is developmentally regulated and correlates to the amount
of neuronal gap junction coupling (Arumugam et al. 2005;
Sohl et al. 1998). Cx36 expression and gap junction coupling
increase in the rat hypothalamus during early postnatal development, peaking on postnatal day 15, and then decrease during
later developmental stages (Arumugam et al. 2005; Gulisano
et al. 2000). Similar changes are observed in hypothalamic
cultures prepared from rats and wild-type mice (Arumugam
et al. 2005). Neuronal gap junction coupling is absent in the
hypothalamus of Cx36 knockout mice (Long et al. 2005). In
our study, neither NMDA receptor blockade nor NMDA receptor hyperactivity induces neuronal cell death in cultures
prepared from Cx36 knockout mice. This together with the fact
that the gap junction blocker carbenoxolone does not have any
additional effects in Cx36 knockout cultures specifically sugJ Neurophysiol • VOL
gest the critical role for Cx36-containing neuronal gap junctions in NMDA receptor– dependent regulation of cell death in
developing neurons. Additionally, hemichannels apparently do
not contribute to NMDA receptor– dependent cell death, as
evident from the dye transfer experiments.
As indicated earlier, an inactivation or hyperactivation of
NMDA receptors does not affect neuronal survival in hypothalamic cultures on DIV1– 4. In neuronal cultures, a significant number of neurons dies during the first several days after
plating (Fig. 1C). This cell death is likely to be due to the
traumatic cell injury caused by the culture preparation procedures. Therefore an alternative explanation for the lack of
NMDA receptor– dependent cell death on DIV1– 4 may be
such that the cell death exists, but is masked by the massive
trauma-related neurodegeneration. However, given that immature neurons are highly tolerant to glutamate-dependent neurotoxicity (Choi et al. 1987) we believe that this possibility is
unlikely.
In our study, the prolonged blockade of NMDA receptors on
DIV4 –DIV31 does not induce neuronal cell death, but, instead,
increases the neuronal survival (Fig. 3C). This suggests an
adaptation of neurons to the chronic change in the level of
NMDA receptor– dependent excitatory activity in a neuronal
network. These results agree with the data reported by Obrietan
and van den Pol (1995) showing that the chronic blockade of
ionotropic glutamate receptors (with AP5 and CNQX) increases neuronal survival in hypothalamic cultures. Our results
in hypothalamic cultures also correlate with the previous observations indicating that inactivation of NMDA receptors in
neuronal cortical cultures induces cell death, but inactivation of
non-NMDA receptors does not have an effect on neuronal
survival (Yoon et al. 2003).
What is the nature of neurodegenerative signals?
Gap junctions are channels between cells that allow the
passage of molecules and ions ⬍1–1.5 kDa (Bennett and Zukin
2004; Simpson et al. 1977). If gap junctions contribute to the
cell death caused by increased or decreased NMDA receptor
function then the critical components that induce (or propagate) such cell death must be able to pass through the gap
junction channels. It is of particular interest that cell death
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
FIG. 5. Connexin 36 (Cx36) knockout prevents neuronal cell death caused by either hypofunction or hyperfunction of NMDA receptors in mice hypothalamic
cultures. A and B: treating wild-type cultures (white bars) with AP5 (A), NMDA (B), or Bic/PiTX (B) on DIV14 –17 induces cell death, which is prevented by
the gap junction blocker CBX. None of the 3 treatments induces neuronal cell death in cultures prepared from Cx36 knockout mice (gray bars). CBX applied
alone does not affect neuronal survival under all conditions. Statistical significance (see the figure legend to Fig. 2) was calculated within the same experimental
groups (i.e., between all wild-type cultures and between all knockout cultures). Sample size: n ⱖ 4 for each test.
2884
DE RIVERO VACCARI, CORRIVEAU, AND BELOUSOV
Functional implications
FIG. 6. Neither activation nor inactivation of NMDA receptors opens
neuronal hemichannels. Sample images (A–D) and statistical analysis (E) from
dye transfer experiments. A and B: calcein fluorescence in neurons before (A)
and 25 min after (B) NMDA application. C and D: calcein fluorescence in
neurons before (C) and 25 min after (D) AP5 application. E: statistical analysis
was performed using paired Student’s t-test. Sample size: n ⫽ 120 neurons
(NMDA test) and n ⫽ 121 neuron (AP5 test). Scale in E represents absolute
fluorescence intensity in arbitrary units (0 – 4,096).
caused in developing neurons by NMDA receptor hypofunction is dependent on Bax/cytochrome c apoptotic mechanisms
(de Rivero Vaccari et al. 2006), whereas NMDA receptor–
dependent excitotoxicity has been reported to be dependent on
poly(ADP-ribose) polymerase and free-radical mechanisms,
and may be Bax independent (Yu et al. 2003). Findings on
baby hamster kidney cells and on Xenopus oocytes suggest that
gap junctions play a role in propagation of death signals
mediated by cytochrome c (Udawatte and Ripps 2005) and that
inositol triphosphate (IP3) and Ca2⫹ likely serve as such gap
junction–permeable signals (Cusato et al. 2006). Additionally,
it has been suggested that Na⫹, Ca2⫹, and IP3 are generated
during NMDA receptor– dependent excitotoxicity and serve as
gap junction–permeable death signals during ischemia and
traumatic injury (Frantseva et al. 2002a,b). A focus of future
studies will be to identify the specific gap junction–permeable
signaling molecules that are generated during the NMDA
J Neurophysiol • VOL
Although gap junction coupling and the expression of Cx36
are high during early postnatal development and increase in the
nervous system during ischemia and traumatic injury (Arumugam et al. 2005; de Pina-Benabou et al. 2005; Gulisano
et al. 2000; Oguro et al. 2001), whether gap junctions contribute to neural cell death or are neuroprotective is controversial
(Perez Velazquez et al. 2003). Some reports indicate that
blocking gap junctions during development, ischemia, or traumatic brain injury rescues cells from apoptosis or necrosis,
suggesting that gap junctions contribute to neural cell death
(Cusato et al. 2003, 2006; de Pina-Benabou et al. 2005;
Frantseva et al. 2002a,b; Nodin et al. 2005; Perez Velazquez
et al. 2006; Rami et al. 2001; Rawanduzy et al. 1997; Udawatte
and Ripps 2005); others demonstrate that gap junctions are
neuroprotective (Blanc et al. 1998; Nakase et al. 2003; Oguro
et al. 2001; Ozog et al. 2002; Siushansian et al. 2001;
Striedinger et al. 2005). Apparently, gap junctions may contribute to both neurodegeneration and neuroprotection depending on the type of connexins, cells coupled, region of the
nervous system, stage of development, type of injury, and other
factors. In our experimental conditions gap junctions are critical for the induction of two different types of NMDA receptor– dependent neuronal cell death.
Ischemic and NMDA mediated toxicity models in vivo show
that the damage is greater in younger than that in adult brains
(Ikonomidou et al. 1989). Farahani and colleagues (2005)
suggested that the abundance of gap junction coupling in the
immature brain is the reason that after hypoxic-ischemic attacks the damage progresses faster in immature than in mature
brains. Our data in cultures correlate with this idea and suggest
that there is a window of opportunity for NMDA receptor–
dependent neurodegenerative signals to kill neurons, which is
during the periods when gap junction coupling is high.
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
receptor hypoactivity and hyperactivity and to establish how
they interact with other factors/pathways that are responsible
for the regulation of cell survival and cell death.
The drug treatments used in this study, including those that
increase or decrease NMDA receptor function, were bathapplied and thus should have affected all neurons in the
cultures. If death signals were generated in all neurons independently and equally, it would be expected that the affected
neurons would die irrespective of whether they are coupled
to other neurons. However, NMDA receptor– dependent cell
death was prevented by inactivation of gap junctions, indicating that the gap junction– coupled neurons have a higher
probability of dying than those that are uncoupled. We thus
hypothesize that the generation of death signals is unequal in
different neurons. This can be due to unequal distribution of
NMDA receptors among different neurons and/or unequal
expression of NMDA receptor– dependent intracellular signaling pathways and molecules. It is possible that at the doses of
NMDA receptor agonist and antagonist that were used, only a
small fraction of neurons generates lethal levels of gap junction–permeable death signals. The blockade of gap junctions
rescues the majority of neurons and only a small (nonsignificant) fraction of neurons dies. We propose that these are the
neurons that produce lethal levels of gap junction–permeable
death signals. Future experiments will evaluate this prediction.
ROLE OF GAP JUNCTIONS IN NMDA RECEPTOR-DEPENDENT CELL DEATH
ACKNOWLEDGMENTS
We thank J. V. Denisova for technical contributions and Drs. Marla Feller
and David Paul for providing connexin 36 knockout mice.
GRANTS
This research was supported by National Institute on Drug Abuse Grant
RO1 DA-015088, National Science Foundation Grant IBN-0117603, American Heart Association Grant 0350530N, and a joint Kansas Institutional
Development Awards Network of Biomedical Research Excellence/University
of Kansas Medical Center grant to A. B. Belousov. Contributions by R.
Corriveau were supported by a Louisiana Board of Regents Research Competitiveness Subprogram and National Institutes of Health Division of Research Resources Grant P20RR-16816.
REFERENCES
Adams SM, de Rivero Vaccari JC, Corriveau RA. Pronounced cell death in
the absence of NMDA receptors in the developing somatosensory thalamus.
J Neurosci 24: 9441–9450, 2004.
Al-Ubaidi MR, White TW, Ripps H, Poras I, Avner P, Gomes D, Bruzzone
R. Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap junctional protein expressed preferentially in retina and brain. J Neurosci Res 59: 813– 826, 2000.
J Neurophysiol • VOL
Arumugam H, Liu X, Colombo PJ, Corriveau RA, Belousov AB. NMDA
receptors regulate developmental gap junction uncoupling via CREB signaling. Nat Neurosci 8: 1720 –1726, 2005.
Arundine M, Tymianski M. Molecular mechanisms of glutamate-dependent
neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci
61: 657– 668, 2004.
Bani-Yaghoub M, Underhill TM, Naus CC. Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal
carcinoma cells. Dev Genet 24: 69 – 81, 1999.
Becker DL, Mobbs P. Connexin alpha1 and cell proliferation in the developing chick retina. Exp Neurol 156: 326 –332, 1999.
Belluardo N, Mudò G, Trovato-Salinaro A, Le Gurun S, Charollais A,
Serre-Beinier V, Amato G, Haefliger JA, Meda P, Condorelli DF.
Expression of connexin36 in the adult and developing rat brain. Brain Res
865: 121–138, 2000.
Belousov AB, O’Hara BF, Denisova JV. Acetylcholine becomes the major
excitatory neurotransmitter in the hypothalamus in vitro in the absence of
glutamate excitation. J Neurosci 21: 2015–2027, 2001.
Bennett MV, Zukin RS. Electrical coupling and neuronal synchronization in
the mammalian brain. Neuron 41: 495–511, 2004.
Biegon A, Fry PA, Paden CM, Alexandrovich A, Tsenter J, Shohami E.
Dynamic changes in N-methyl-D-aspartate receptors after closed head injury
in mice: implications for treatment of neurological and cognitive deficits.
Proc Natl Acad Sci USA 101: 5117–5122, 2004.
Blanc EM, Bruce-Keller AJ, Mattson MP. Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced
disruption of Ca2⫹ homeostasis and cell death. J Neurochem 70: 958 –970,
1998.
Buss RR, Oppenheim RW. Role of programmed cell death in normal
neuronal development and function. Anat Sci Int 79: 191–197, 2004.
Chang Q, Pereda A, Pinter MJ, Balice-Gordon RJ. Nerve injury induces
gap junctional coupling among axotomized adult motor neurons. J Neurosci
20: 674 – 684, 2000.
Choi DW. Ischemia-induced neuronal apoptosis. Curr Opin Neurobiol 6:
667– 672, 1996.
Choi DW, Maulucci-Gedde M, Kriegstein AR. Glutamate neurotoxicity in
cortical cell culture. J Neurosci 7: 357–368, 1987.
Cole K, Perez-Polo JR. Neuronal trauma model: in search of Thanatos. Int J
Dev Neurosci 22: 485– 496, 2004.
Connors BW, Benardo LS, Prince DA. Coupling between neurons of the
developing rat neocortex. J Neurosci 3: 773–782, 1983.
Contestabile A. Roles of NMDA receptor activity and nitric oxide production
in brain development. Brain Res Brain Res Rev 32: 476 –509, 2000.
Cusato K, Bosco A, Rozental R, Guimaraes CA, Reese BE, Linden R,
Spray DC. Gap junctions mediate bystander cell death in developing retina.
J Neurosci 23: 6413– 6422, 2003.
Cusato K, Ripps H, Zakevicius J, Spray DC. Gap junctions remain open
during cytochrome c-induced cell death: relationship of conductance to
“bystander” cell killing. Cell Death Differ 13: 1707–1714, 2006.
Davis EC, Popper P, Gorski RA. The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain Res
734: 10 –18, 1996.
Deans MR, Gibson JR, Sellitto C, Connors BW, Paul DL. Synchronous
activity of inhibitory networks in neocortex requires electrical synapses
containing connexin36. Neuron 31: 477– 485, 2001.
de Pina-Benabou MH, Szostak V, Kyrozis A, Rempe D, Uziel D, UrbanMaldonado M, Benabou S, Spray DC, Federoff HJ, Stanton PK,
Rozental R. Blockade of gap junctions in vivo provides neuroprotection
after perinatal global ischemia. Stroke 36: 2232–2237, 2005.
de Rivero Vaccari JC, Casey GP, Aleem S, Park WM, Corriveau RA.
NMDA receptors promote survival in somatosensory relay nuclei by inhibiting Bax-dependent developmental cell death. Proc Natl Acad Sci USA 103:
16971–16976, 2006.
De Vuyst E, Decrock E, Cabooter L, Dubyak GR, Naus CC, Evans WH,
Leybaert L. Intracellular calcium changes trigger connexin 32 hemichannel
opening. EMBO J 25: 34 – 44, 2006.
Farahani R, Pina-Benabou MH, Kyrozis A, Siddiq A, Barradas PC, Chiu
FC, Cavalcante LA, Lai JC, Stanton PK, Rozental R. Alterations in
metabolism and gap junction expression may determine the role of astrocytes as “good Samaritans” or executioners. Glia 50: 351–361, 2005.
Frantseva MV, Kokarovtseva L, Naus CG, Carlen PL, MacFabe D, Perez
Velazquez JL. Specific gap junctions enhance the neuronal vulnerability to
brain traumatic injury. J Neurosci 22: 644 – 653, 2002a.
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
In neuronal cultures prepared from embryonic day 18 –19
rats, DIV14 –17 approximately correlates with the 1st to 2nd
wk of postnatal development in vivo (given some delay in the
development caused by the cell culture preparation). This time
in vivo in the hypothalamus is the period of naturally occurring
cell death (Davis et al. 1996; Yoshida et al. 2000) and when the
gap junction coupling peaks (Arumugam et al. 2005). In
in vitro conditions, this also is the period when NMDA receptor hypoactivity and hyperactivity induce neuronal cell death
(present results). We hypothesize that the contribution of gap
junctions in the NMDA receptor– dependent cell death that is
revealed in this study in cultures on DIV14 –17, may reflect
two possibilities. The first possibility is that gap junctions and
NMDA receptors work in concert to regulate survival versus
cell death decisions during neuronal development and, as a
result, they contribute to the regulation of the hypothalamic
network development. If this is the case, then the “critical
period” for NMDA receptor– dependent neurodegenerative
signals to kill neurons exists during the short period of postnatal development—i.e., at the time when neuronal gap junction coupling is high. During this time, NMDA receptors
regulate cell death by gap junctions, whether the cell death is
induced by increased or decreased NMDA receptor function.
As development proceeds to later stages, NMDA receptors
down-regulate neuronal gap junctions (Arumugam et al. 2005).
It is possible that the cooperation of NMDA receptors and gap
junctions depends on cofactors that determine the timing and
amount of gap junction up- and down-regulation. In the mature
nervous system, traumatic injury and ischemia may reactivate
neuronal susceptibility by increasing gap junction coupling to
higher levels than normally found among mature neurons. The
second possibility is that the presence of gap junctions simply
provides the general channel for propagation of death/survival
signals independently of the nature of these signals. In this case,
the signals will be propagated at any time when the gap junction
coupling is high. A better understanding of how gap junctions and
NMDA receptors interact in the regulation of cell death/survival
will allow for the development of treatments that safeguard
neuronal survival following traumatic injuries, ischemic attacks,
or exposure to teratogens that disrupt NMDA receptor function.
2885
2886
DE RIVERO VACCARI, CORRIVEAU, AND BELOUSOV
J Neurophysiol • VOL
Peinado A, Yuste R, Katz LC. Extensive dye coupling between rat neocortical neurons during the period of circuit formation. Neuron 10: 103–114,
1993.
Perez Velazquez JL, Frantseva MV, Naus CC. Gap junctions and neuronal
injury: protectants or executioners? Neuroscientist 9: 5–9, 2003.
Perez Velazquez JL, Kokarovtseva L, Sarbaziha R, Jeyapalan Z, Leshchenko Y. Role of gap junctional coupling in astrocytic networks in the
determination of global ischaemia-induced oxidative stress and hippocampal
damage. Eur J Neurosci 23: 1–10, 2006.
Rami A, Volkmann T, Winckler J. Effective reduction of neuronal death by
inhibiting gap junctional intercellular communication in a rodent model of
global transient cerebral ischemia. Exp Neurol 170: 297–304, 2001.
Rash JE, Staines WA, Yasumura T, Patel D, Furman CS, Stelmack GL,
Nagy JI. Immunogold evidence that neuronal gap junctions in adult rat brain
and spinal cord contain connexin-36 but not connexin-32 or connexin-43.
Proc Natl Acad Sci USA 97: 7573–7578, 2000.
Rawanduzy A, Hansen A, Hansen TW, Nedergaard M. Effective reduction
of infarct volume by gap junction blockade in a rodent model of stroke.
J Neurosurg 87: 916 –920, 1997.
Scemes E, Spray DC. Increased intercellular communication in mouse astrocytes exposed to hyposmotic shocks. Glia 24: 74 – 84, 1998.
Scheetz AJ, Constantine-Paton M. Modulation of NMDA receptor function:
implications for vertebrate neural development. FASEB J 8: 745–752, 1994.
Simpson I, Rose B, Loewenstein WR. Size limit of molecules permeating the
junctional membrane channels. Science 195: 294 –296, 1977.
Siushansian R, Bechberger JF, Cechetto DF, Hachinski VC, Naus CC.
Connexin43 null mutation increases infarct size after stroke. J Comp Neurol
440: 387–394, 2001.
Sohl G, Degen J, Teubner B, Willecke K. The murine gap junction gene
connexin36 is highly expressed in mouse retina and regulated during brain
development. FEBS Lett 428: 27–31, 1998.
Striedinger K, Petrasch-Parwez E, Zoidl G, Napirei M, Meier C, Eysel
UT, Dermietzel R. Loss of connexin36 increases retinal cell vulnerability to
secondary cell loss. Eur J Neurosci 22: 605– 616, 2005.
Thompson RJ, Zhou N, MacVicar BA. Ischemia opens neuronal gap junction hemichannels. Science 312: 924 –927, 2006.
Udawatte C, Ripps H. The spread of apoptosis through gap junctional
channels in BHK cells transfected with Cx32. Apoptosis 10: 1019 –1029,
2005.
Valiunas V, Mui R, McLachlan E, Valdimarsson G, Brink PR, White TW.
Biophysical characterization of zebrafish connexin35 hemichannels. Am J
Physiol Cell Physiol 287: C1596 –C1604, 2004.
Waxman EA, Lynch DR. N-methyl-D-aspartate receptor subtypes: multiple
roles in excitotoxicity and neurological disease. Neuroscientist 11: 37– 49,
2005.
Yoon WJ, Won SJ, Ryu BR, Gwag BJ. Blockade of ionotropic glutamate
receptors produces neuronal apoptosis through the Bax-cytochrome
C-caspase pathway: the causative role of Ca2⫹ deficiency. J Neurochem 85:
525–533, 2003.
Yoshida M, Yuri K, Kizaki Z, Sawada T, Kawata M. The distributions of
apoptotic cells in the medial preoptic areas of male and female neonatal rats.
Neurosci Res 36: 1–7, 2000.
Yu SW, Wang H, Dawson TM, Dawson VL. Poly(ADP-ribose) polymerase-1 and apoptosis inducing factor in neurotoxicity. Neurobiol Dis 14:
303–317, 2003.
98 • NOVEMBER 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 18, 2017
Frantseva MV, Kokarovtseva L, Perez Velazquez JL. Ischemia-induced
brain damage depends on specific gap junctional coupling. J Cereb Blood
Flow Metab 22: 453– 462, 2002b.
Giza CC, Maria NS, Hovda DA. N-methyl-D-aspartate receptor subunit
changes after traumatic injury to the developing brain. J Neurotrauma 23:
950 –961, 2006.
Goodman CS, Shatz CJ. Developmental mechanisms that generate precise
patterns of neuronal connectivity. Cell Suppl 72: 77–98, 1993.
Gulisano M, Parenti R, Spinella F, Cicirata F. Cx36 is dynamically
expressed during early development of mouse brain and nervous system.
Neuroreport 11: 3823–3828, 2000.
Hardingham GE, Bading H. Coupling of extrasynaptic NMDA receptors to
a CREB shut-off pathway is developmentally regulated. Biochim Biophys
Acta 1600: 148 –153, 2002.
Hardingham GE, Bading H. The Yin and Yang of NMDA receptor signalling. Trends Neurosci 26: 81– 89, 2003.
Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K,
Tenkova TI, Stefovska V, Turski L, Olney JW. Blockade of NMDA
receptors and apoptotic neurodegeneration in the developing brain. Science
283: 70 –74, 1999.
Ikonomidou C, Mosinger JL, Salles KS, Labruyere J, Olney JW. Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels
sensitivity to N-methyl-D-aspartate neurotoxicity. J Neurosci 9: 2809 –2818,
1989.
Kandler K, Katz LC. Neuronal coupling and uncoupling in the developing
nervous system. Curr Opin Neurobiol 5: 98 –105, 1995.
Long MA, Jutras MJ, Connors BW, Burwell RD. Electrical synapses
coordinate activity in the suprachiasmatic nucleus. Nat Neurosci 8: 61– 66,
2005.
Lo Turco JJ, Kriegstein AR. Clusters of coupled neuroblasts in embryonic
neocortex. Science 252: 563–566, 1991.
Nakase T, Fushiki S, Naus CC. Astrocytic gap junctions composed of
connexin 43 reduce apoptotic neuronal damage in cerebral ischemia. Stroke
34: 1987–1993, 2003.
Nakazawa K, McHugh TJ, Wilson MA, Tonegawa S. NMDA receptors,
place cells and hippocampal spatial memory. Nat Rev Neurosci 5: 361–372,
2004.
Nodin C, Nilsson M, Blomstrand F. Gap junction blockage limits intercellular spreading of astrocytic apoptosis induced by metabolic depression.
J Neurochem 94: 1111–1123, 2005.
Obrietan K, Van den Pol AN. Calcium hyperexcitability in neurons cultured
with glutamate receptor blockade. J Neurophysiol 73: 1524 –1536, 1995.
Oguro K, Jover T, Tanaka H, Lin Y, Kojima T, Oguro N, Grooms SY,
Bennett MV, Zukin RS. Global ischemia-induced increases in the gap
junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and
enhanced vulnerability of Cx32 knock-out mice. J Neurosci 21: 7534 –7542,
2001.
Ozog MA, Siushansian R, Naus CC. Blocked gap junctional coupling
increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures.
J Neuropathol Exp Neurol 61: 132–141, 2002.
Parson SH, Price JF, Ribchester RR. Cell viability and laminin-induced
neurite outgrowth in cultures of embryonic chick neural tube cells: effects of
cytosine-␤-D-arabinofuranoside. Neurodegeneration 4: 99 –106, 1995.