Expression of connexin genes in hippocampus of kainate-treated and kindled rats under conditions of experimental epilepsy
ABSTRACT We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e., animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats only moderate increases of GFAP mRNA and protein were detected 2–3 weeks (group 2) or 4–6 weeks (group 1) after the last stage 5 induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43, Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins was indistinguishable from control animals.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Gap junctions are intercellular membrane channels that provide direct cytoplasmic continuity between adjacent cells. This communication can be affected by changes in expression of gap junctional subunits called Connexins (Cx). Changes in the expression and function of connexins are associated with number of brain neurodegenerative diseases. Neuroinflammation is a hallmark of various central nervous system (CNS) diseases, like multiple sclerosis, Alzheimer's disease and epilepsy. Neuroinflammation causes change in Connexins expression. Hippocampus, one of the main brain regions with a wide network of Gap junctions between different neural cell types, has particular vulnerability to damage and consequent inflammation. Cx32 - among Connexins- is expressed in hippocampal Olygodandrocytes and some neural subpopulations. Although multiple lines of evidence indicate that there is an association between neuroinflammation and the expression of connexin, the direct effect of neuroinflammation on the expression of connexins has not been well studied. In the present study, the effect of neuroinflammation induced by the Lipopolysaccharide (LPS) on Cx32 gene and protein expressions in rat hippocampus is evaluated.Autonomic neuroscience: basic & clinical 01/2013; 4(4):334-40. · 1.82 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Gap junction (GJ) channels have been recognized as an important mechanism for synchronizing neuronal networks. Herein, we investigated the participation of GJ channels in the pilocarpine-induced status epilepticus (SE) by analyzing electrophysiological activity following the blockade of connexins (Cx)-mediated communication. In addition, we examined the regulation of gene expression, protein levels, phosphorylation profile and distribution of neuronal Cx36, Cx45 and glial Cx43 in the rat hippocampus during the acute and latent periods. Electrophysiological recordings revealed that the GJ blockade anticipates the occurrence of low voltage oscillations and promotes a marked reduction of power in all analyzed frequencies.Cx36 gene expression and protein levels remained stable in acute and latent periods, whereas upregulation of Cx45 gene expression and protein redistribution were detected in the latent period. We also observed upregulation of Cx43 mRNA levels followed by changes in the phosphorylation profile and protein accumulation. Taken together, our results indisputably revealed that GJ communication participates in the epileptiform activity induced by pilocarpine. Moreover, considering that specific Cxs undergo alterations through acute and latent periods, this study indicates that the control of GJ communication may represent a focus in reliable anti-epileptogenic strategies. Citation: Kinjo ER, Higa GSV, Morya E, Valle AC, Kihara AH, et al. (2014) Reciprocal Regulation of Epileptiform Neuronal Oscillations and Electrical Synapses in the Rat Hippocampus. PLoS ONE 9(10): e109149. doi:10.1371/journal.pone.0109149 Copyright: ß 2014 Kinjo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the following funders: FAPESP and CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal's policy and have the following conflict: AHK is part of PLOS ONE Academic Board.PLoS ONE 10/2014; 9(10). · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Enhanced gap junctional communication (GJC) between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other non-specific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co(++) seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43) is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45), and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions (GJs), diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by traditional gap junctional pharmacological blockers. Although there is no doubt that connexin-based GJs and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to be elucidated.Frontiers in Physiology 05/2014; 5:172.
Molecular Brain Research 83 (2000) 44–51
Expression of connexin genes in hippocampus of kainate-treated and
kindled rats under conditions of experimental epilepsy
Goran Sohl , Martin Guldenagel , Heinz Beck , Barbara Teubner , Otto Traub ,
Rafael Gutierrez , Uwe Heinemann , Klaus Willecke
Abteilung fur Molekulargenetik, Institut fur Genetik, Universitat Bonn, 53117 Bonn, Germany
Klinik fur Epileptologie, Universitat Bonn, 53117 Bonn, Germany
Institut fur Physiologie, Charite Berlin, Germany
Accepted 2 August 2000
We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e.,
animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular
location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in
kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased
expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats
only moderate increases of GFAP mRNA and protein were detected 2–3 weeks (group 2) or 4–6 weeks (group 1) after the last stage 5
induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were
nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein
was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or
unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43,
Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins
was indistinguishable from control animals.
2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Epilepsy: human studies and animal models
Keywords: Gap junction; Connexin; Spatial buffering
providing a pathway for both electrical and chemical
communication between coupled cells . So far, 15
different connexin genes have been described in the
murine genome [21,32]. Only some were demonstrated to
be expressed in specific cell types of rodent and human
brain: Cx43 is expressed in astrocytes [8,45], whereas
Cx32 was detected in oligodendrocytes and in some
neuronal subpopulations . Recent results showed that
Cx36 is expressed in the inferior olive and in hippocampal
neurons of the CA3 region , while Cx30 is co-expressed
with Cx43 in adult astrocytes [18,22].
Hypersynchronized neuronal discharges, which are
characteristic of recurrent electrical field burst activity of
an epileptic focus, were attributed to gap junctional
communication in hippocampal slices during induction of
Gap junctions are intercellular transmembrane channels,
which facilitate ionic homeostasis and synchronization of
action potentials in the nervous system [8,28]. The aqueous
pores of gap junction channels are formed by docking of
two hemichannels, each comprised of six connexin (Cx)
protein subunits that permit the passage of ions, metabo-
lites and second messenger molecules (,1 kDa), thereby
GFAP, glial fibrillary acidic protein; i.p., intraperitoneal
*Corresponding author. Tel.: 149-228-734-210; fax: 149-228-734-
E-mail address: email@example.com (K. Willecke).
0169-328X/00/$ – see front matter
2000 Elsevier Science B.V. All rights reserved.
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51 45
field burst activity in calcium-free medium, when synaptic
transmission was blocked [17,31,41]. Axo-axonal gap
junctions between pyramidal neurons were suggested to
influence at least high-frequency (125–333 Hz) network
oscillations superimposed on epileptiform field potentials
[10,40]. A recent study revealed, that neuronal acidifica-
tion was able to shorten epileptiform activity in a slice
model where synaptic transmission was omitted .
Astroglial coupling via Cx43 containing gap junctions is
thought to provide ionic homeostasis and spatial buffering
of potassium ions, which was shown to be modulated by
neurotransmitters or intracellular signalling . Gap
junction channels in astrocytes could also mediate the
propagation of calcium waves [4,7] and influence neuronal
activity [24,43]. For this reason, the expression of astrogli-
al Cx43 has been studied in human and experimental
epilepsy. Elisevich et al.  used control tissues from
nonepileptic patients who underwent temporal lobectomy.
In addition, specific electroencephalographic criteria were
used to identify epileptogenicity within the anterior hip-
pocampus, which was taken for combined Northern and
immunoblot analysis of Cx43. In contrast to Naus et al.
, who described a significant 3-fold elevated level of
Cx43 mRNA in samples of epileptic temporal lobe tissues,
Elisevich et al.  found no upregulation of Cx43 in the
presence of focal epileptogenicity. These authors con-
cluded, that the clinical outcome was independent of the
Cx43 mRNA or protein content. Moreover, Elisevich et al.
[12,13] extended their Cx43 expression studies to kindled
and tetanus toxin-treated rats. Left amygdalas were kindled
and tetanus toxin injected, respectively, while surgical
controls were used with implanted electrodes or canules.
When, after 4 weeks recovery, the left amygdala of each
animal was used for combined Northern and immuno-
fluorescence analyses, both kinds of rats showed no
significant Cx43 alterations, despite varying degrees of
epileptogenicity. Only in kainate-treated rats, however,
Khurgel and Ivy  found a dramatic decrease of short-
term (48 h) Cx43 mRNA expression, which could not be
detected in kindled rats.
Here, we have analyzed kainate-treated and kindled rats
with regard to hippocampal expression of Cx43, 32, 30 and
36, i.e., connexins, whose expression might be altered
recurrent ‘wet dog shakes’ and rearing, followed by
bilateral forelimb clonus and occasional falling and jump-
ing as described byVeliskova et al. . Age-matched rats,
injected with saline only were prepared as control group.
Thirty days after the last kainate administration all animals
were decapitated under deep ether anesthesia.
2.2. Kindling of rats
Adult male Wistar rats (83–95 days; 230–250 g) were
used. Bipolar stainless steel electrodes (80 kV) were
stereotactically implanted under ketamine anesthesia (60
mg/kg body weight) into the left basolateral amygdala (AP
2.5; L 5; H 8.5; Paxinos and Watson ). After a
postsurgical recovery period of 7–10 days, six of the
animals were stimulated daily through the implanted
electrode with a train of pulses lasting 0.1 ms at 60 Hz for
2 s. The intensity used for stimulation was set to induce
initially only a twitching of the eye ipsilateral to the
stimulated amygdala. The behavioral changes induced by
the kindling process were scored according to the be-
havioral scale of Racine . Four unstimulated animals
were used as a control group. Rats were kindled, until they
had six consecutive stage 5 seizures. Two to three weeks
(group 2) and 4–6 weeks (group 1) after the last seizure,
the animals were decapitated under deep ether anesthesia.
The brains of kainate-treated or kindled rats were
collected in ice-cold phosphate-buffered saline (PBS ).
Then left and right hippocampi were dissected and divided
for subsequent Northern blot, immunoblot and immuno-
fluorescence analyses. Specimens for Northern blot hybrid-
ization were immediately frozen in liquid nitrogen, while
tissue samples for immunoblot and immunofluorescence
analyses as well as Nissl stains were frozen on dry ice.
All experiments involving kainate treatment and kindl-
ing of rats were carried out with permission of local
authorities and in accordance with the German law for
2.3. Northern blot analysis
Total RNA from both left and right hippocampal
sections was prepared with TRIzol -reagent according to
the manufacturer (Gibco-BRL, Eggenstein, Germany).
RNA (20 mg each) was electrophoresed  and trans-
ferred to Hybond-N nylon membrane (Amersham Interna-
tional, Amersham, Bucks, UK) by capillary diffusion in
203 standard saline citrate (SSC). Transcript sizes were
determined by comparison to an RNA ladder of stan-
dardized molecular mass (Gibco-BRL). The blots were
probed with the following random primed
fragments (multiprime labelling kit, Amersham) of the
respective rodent connexin coding DNA and a 1.2-kb
HindIII–XbaI cDNA fragment of the gene for the glial
fibrillary acidic protein (GFAP) : mouse Cx30 KpnI
fragment (0.6 kb) ; rat Cx32 EcoRI fragment (1.5 kb)
2. Material and methods
2.1. Kainate treatment of rats
Young adult male Sprague–Dawley rats (26–33 days;
60–100 g) were i.p. injected twice with kainate (12.5
mg/kg body weight) on two consecutive days. All animals
were observed for 4–6 h after injection. Treated rats
showed an onset of seizure activity 15–20 min after
kainate administration. Seizures were characterized by
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51
mRNA expression of connexins and GFAP in hippocampal tissue of
kainate-treated and kindled rats
; rat Cx36 PstI fragment (1.1 kb) ; and rat Cx43
HindIII–StuI fragment (1.4 kb) . DNA probes of
specific activities between 0.5 and 1.0310 cpm/mg DNA
were added to fresh prehybridization solution at 5310
cpm/ml. Hybridization was carried out under high string-
ency conditions (55% formamide, 53 SSC, 53 Denhardt’s
and 0.5% sodium dodecyl sulfate (SDS)) in the presence of
60 mg/ml heat-denatured salmon sperm DNA (Boehringer,
Mannheim, Germany) for 16 h at 428C . After the last
washing step with 0.13 SSC/0.1% SDS for 10 min at
658C, nylon membranes were sealed in plastic wrap and
exposed to Kodak XAR X-ray film (Eastman Kodak,
Rochester, NY, USA) with intensifying screen at 2708C
for 3–4 weeks. The amounts of total RNA on Northern
blots were normalized by hybridization to a 1.2-kb frag-
ment of human glyceraldehydephosphate dehydrogenase
(GAPDH) . Subsequently, the same Northern blot
membrane was hybridized three times, simultaneously with
two probes. Densitometric analyses were performed with
the Image Master Package (Pharmacia, San Francisco, CA,
aSummary of densitometric values obtained from Northern hybridization
in Fig. 1. The results are presented as percent in each category using
mean values6standard error.
Asterisks indicate significance.
samples were incubated with secondary antibodies (TRI-
TC-conjugated anti-rabbit IgG, Dianova, Hamburg, Ger-
many) diluted 1:500 in blocking reagent for 2 h. As
negative control, specimen were incubated with blocking
reagent without primary antibodies. After incubation,
sections were washed in PBS , mounted on glass slides in
glycerol containing 2.5% KI and 7.5% PBS
darkness at 48C. Fluorescent signals were documented on
Ilford HP 5 film using a Zeiss Axiophot photomicroscope
equipped with a 363 objective and appropriate filters.
and stored in
2.4. Immunoblot analysis
Inpreparation forsodium dodecylsulfate–poly-
acrylamide gel electrophoresis (SDS–PAGE), both left and
right hippocampal sections as well as control tissues were
pulverized under liquid nitrogen and vacuum dried. Elec-
trophoresis and electroblotting were carried out according
to Ref. . The following antibodies were used: rabbit
polyclonal anti-Cx30 ; rabbit polyclonal anti-Cx32
; rabbit polyclonal anti-Cx36 ; rabbit polyclonal
anti-Cx43 amino acids 360–382 ; and mouse mono-
clonal anti-GFAP (Sigma G-3893). Antibodies were di-
luted in 1xRotiBlock(Roth, Karlsruhe, Germany), incu-
bated overnight with blocked membranes at room tempera-
ture during slight agitation, followed by washing in
1xRotiBlock . Immunocomplexes were analyzed using
125I-labeled protein A. GFAP immunoblots were analyzed
using the secondary antibody horseradish peroxidase-
conjugated goat anti-mouse Ig of the ECL -detection kit
(Amersham) according to the manufacturer. Bands were
visualized using autoradiography as described for Northern
2.6. Data analysis
The densitometric values (d.v.) obtained after Northern
blot hybridization for both the tested connexins and GFAP
were normalized to the densitometric values of the corre-
sponding GAPDH hybridization. After immunoblotting,
the densitometric values obtained for each connexin and
GFAP were normalized to the densitometric values of the
corresponding Ponceau-S stained protein fractions (data
not shown). Values of the kainate group, kindling group 1
and 2 and their corresponding control groups were ana-
lyzed by the Wilcoxon, Mann and Whitney test and
presented as mean densitometric value6standard error
(Tables 1 and 2). The error-probability of P,0.05 was
used to determine significances indicated by asterisks (*).
Protein expression of connexins and GFAP in hippocampal tissue of
kainate-treated and kindled rats
2.5. Immunofluorescence analysis
Cryostat sections (10 mm) of the middle segment of
each hippocampus were fixed in absolute ethanol (2208C)
for 10 min and then processed at room temperature for
immunofluorescence analysis: the sections were washed in
PBSand pre-incubated for 30 min in blocking reagent
(PBScontaining 4% BSA and 0.1% Triton X-100). For
detection of connexin proteins, sections were incubated in
blocking reagent with affinity-purified anti-Cx43, 36 and
30 antibodies for 1 h. After several washings in PBS ,
aSummary of densitometric values obtained from immunoblot in Fig. 2.
The results are presented as percent in each category using mean
Asterisks indicate significance.
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–5147
expression in the affected hippocampi, we sacrificed all
animals in both kinds of rats after the latent period
described. Relative amounts of mRNAs were determined
by densitometric scans of the autoradiographs shown in
Fig. 1. For kainate-treated rats 5 and 7–10, the mean
densitometric value of GFAP mRNA expression is sig-
nificantly elevated (200645%) compared to 10069% of
control animals 1–3. Accordingly, the mean expression
levels of the four tested connexins are summed up in Table
1. The increased expression of GFAP mRNA demonstrates
reactive gliosis but expression of Cx30 and Cx43 mRNA is
nearly unaltered, although both connexins are expressed in
astrocytes. Interestingly, Cx36 mRNA expression, proven
to occur in hippocampal neurons [1,3] is significantly
reduced (56614%) compared to control (100624%). No
alterations in mRNA expression of Cx32 were determined.
The first kindling group, after 4–6 weeks of recovery,
showed a mean densitometric value of 146659% and the
second group, after 2–3 weeks of recovery,exhibited a
mean value of 1886108% for GFAP mRNA, compared to
100613% of control animals. This indicates no significant
gliosis in these kindled rats  (Fig. 1a). Results of both
kindling groups show slight reduction of Cx43 (7265 and
57613%) and Cx30 (90616 and 74626%) mRNA ex-
pression levels and stronger decrease of Cx36 (56631 and
53623%) expression, of which group 2 (2–3 weeks latent
period) is statistically significant. Concerning Cx32 expres-
sion, again no significant alterations could be detected
when compared to control.
3.1. Kainate-treated rats
Ten rats were used for this experiment. Rats 5 and 7–10
showed bouts of generalized tonic–clonic seizures after the
second kainate injection, lasting for 1–2 h. After a latent
period of 30 days, rats 7–10 exhibited spontaneous sei-
zures. Since animals 4* and 6* showed no major tonic–
clonic seizures, they were excluded from subsequent
evaluations. Animals 1–3 had been mock injected with
3.2. Kindled rats
Thirteen rats were used for this experiment. Animals
were stimulated until they had six consecutive stage 5
seizures. After a latency period of 4–6 weeks, animals 5–9
were sacrificed (kindling group 1), while animals 10–13
with a latent period of 2–3 weeks were combined to form
kindling group 2. Rats 1–4 represent unstimulated control
animals, which also carried implanted electrodes.
3.3. Northern blot analysis
We have evaluated the transcript levels of Cx43, 36, 32
and 30 in both kainate-treated and kindled rats. Since we
were interested in long-lasting effects on connexin gene
Fig. 1. Northern blot hybridization of total RNA (20 mg each) from both left and right hippocampal segments. Blots were first hybridized simultaneously
to pooled DNA probes, coding for the rat Cx32 and 36 genes. After 4 weeks of exposure, blots were stored in 23 SSC for additional 16 weeks to allow for
10 halflife times ofP and rehybridized to two DNA probes coding for the rat Cx43 and mouse Cx30 gene. Finally, blots were rehybridized to DNA
probes coding for human glyceraldehydephosphate dehydrogenase (GAPDH) and mouse glial fibrillary acidic protein (GFAP). The GAPDH hybridization
signals indicate that about equal amounts of total RNA were electrophoresed and blotted. Left panel: kainate-treated rats 4 to 10 were studied after 4 weeks
of recovery. Control animals 1–3 were injected with saline only. Rats 4* and 6* demonstrated no major tonic–clonic seizures and were therefore excluded
from further evaluations. Right panel: kindled rats 5–13 were divided into two subgroups depending on their recovery period. Group 1: rat 5, 6 weeks; rat
6, 4 weeks; rat 7, 4 weeks; rat 8, 4 weeks; rat 9, 4 weeks. Group 2: rat 10, 2 weeks; rat 11, 3 weeks; rat 12, 2 weeks; rat 13, 2 weeks. Control animals 1–4
carried implanted electrodes but were left unstimulated. The results of densitometric evaluation are listed in Tables 1 and 2.
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51
3.4. Immunoblot analysis
3.5. Immunofluorescence analysis
In order to recognize elevated levels of glial connexin
proteins due to astrogliosis, we determined the level of
glial fibrillary acidic protein (GFAP). The expression level
of Cx32 protein or the sensitivity of the corresponding
antibodies were too low to detect this protein unequivo-
cally and so it was omitted from further analyses. Lysates
of Cx43, 36 and 30 transfected HeLa cells [38,37,18] were
used as positive controls (Fig. 2). According to the
densitometric evaluation of the Northern blot hybridiza-
tions, we evaluated the mean GFAP protein abundance for
kainate-treated rats, groups 1 and 2 of kindled rats and the
corresponding controls. Samples from kainate-treated rats
exhibited a GFAP protein content of 278669% compared
to control 100629% (Table 2). The significant increase of
178% in the GFAP protein content after kainate adminis-
tration coincides with the significant increase of the GFAP
mRNA abundance and thus confirms reactive gliosis in this
epilepsy model . Under these conditions protein abun-
dance of Cx30 and Cx43, expressed in astrocytes is nearly
unaltered similar to their mRNA abundances (Table 1).
Even protein content of Cx36 is only slightly decreased
(17%). In both kindling groups, elevated levels of GFAP
protein content might result from its increased transcript
abundancies (Table 1), but do not indicate significant
gliosis in this epilepsy model. Protein content of Cx43 and
Cx30 expressed in astrocytes is unaltered in both groups
(Table 2), whereas Cx36 protein abundance is significantly
decreased (45%) in kindling group 2 (2–3 weeks), pre-
sumably reflecting the significant decrease (47%) of Cx36
mRNA (Table 1). However, Cx36 protein abundance in
kindling group 1 (4–6 weeks), which had more time to
recover after kindling, appeared to be similar to control
Since the function of gap junction-mediated intercellular
communication could also be influenced by the cellular
location of the connexin proteins, we analyzed Cx43, 36
and 30 protein within the CA3 hippocampal region. In this
subregion, moderate neuronal loss was detected by Nissl
stainings in kainate-treated but not in kindled rats (data not
shown). Neither in kainate-treated nor in kindling groups,
any abnormal location of the Cx43, 36 and 30 protein was
seen, compared to the corresponding control sections. Fig.
3 demonstrates in both epileptic rats the location of Cx43
and Cx30 within stratum moleculare, pyramidale and
oriens of the CA3 region, showing the same pattern and
distribution as in corresponding control sections. In these
layers, astrocytic cell bodies and processes are pre-
sent.Thus, at least in kainate-treated rats, Cx43 protein
re-occurred on the astrocytic cell surface after its inter-
mediate internalization and redistribution in the sprouting
filopodia of hypertrophic astrocytes within 1 week after the
last kainate administration . Cx36, mainly expressed in
the pyramidal cell layer of the hippocampal CA3 region
[1,3,35], was not affected, either in location or in abun-
dance, in both kinds of epileptic rats, after 4 weeks of
In this study we have compared mRNA expression,
protein abundance and localization of Cx43, 32, 30 and 36
in kainate-treated and kindled rats, which represent animal
models of human temporal lobe epilepsy, since they mimic
the long-term seizure-prone state known from epileptic
patients. We have addressed the following question: can
Fig. 2. Immunoblot of protein lysates (50 mg each) from left and right hippocampal segments were electrophoresed and blotted. Ponceau-S staining of
membranes confirmed that nearly equal amounts of protein had been transferred. Incubation with antibodies to mouse glial fibrillary acidic protein (GFAP)
was used to evaluate the extent of astrogliosis. Binding of connexin specific antibodies was detected with
and right panel were the same as described in the legend to Fig. 1. In lane H, the lysate of the corresponding HeLa connexin transfectants was
electrophoresed to demonstrate antibody specificity. The results of the densitometric evaluation are listed in Tables 1 and 2.
I-labelled protein A. The samples of the left
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51 49
Fig. 3. Location of Cx43, 36 and 30 protein by indirect immunofluorescence in the CA3-hippocampal subregion of kainate-treated, kindled rats and control
animals, 4 weeks after the end of treatment. Incubation with connexin-specific polyclonal rabbit antibodies and FITC-conjugated goat anti-rabbit IgG is
described in Section 2. The two band-like arrays of Cx43 and Cx30 immunosignals indicate astrocytes arranged on both sides of the pyramidal cell layer, in
which mainly Cx36 signals are visible. No abnormal localization of these connexins is evident in both epilepsy models, when compared to their respective
controls. O, stratum oriens; P, stratum pyramidale; R, stratum radiatum; LM, stratum lacunosum moleculare; M, stratum moleculare; AD, area dentata; H,
long-term aberrant connexin mRNA or protein expression,
induced in chronic epileptogenic tissue, constitute a pos-
sible basis for impaired spatial buffering or for an abnor-
mal neuronal synchronization? We therefore investigated
kainate-treated rats 4 weeks and kindled animals 2–6
weeks after recovery. Despite a significant increase of
GFAP mRNA and protein content due to reactive gliosis in
the kainate-treated rats, no upregulation of astrocytic gap
junction proteins (Cx30 and Cx43) could be documented.
But the neuronal Cx36 mRNA expression in these rats was
significantly decreased (44%), although its protein expres-
sion remained unaltered. In kindled rats, a significant
decline of Cx36 mRNA and protein abundance (47 and
45%) was only seen in group 2, comprising animals with
2–3 weeks recovery after the last stage 5 seizure. In
kindling group 1, however, in which animals recovered for
4–6 weeks, downregulation of Cx36 mRNA and protein
abundance was not significant. No significant alterations
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51
could be documented for astroglial GFAP, Cx30 and Cx43
expression in kainate-treated rats. Under these experimen-
tal conditions, it seems that despite significant astrogliosis
in kainate-treated rats and some tendency towards gliosis
in kindled rats [5,36], the increased number of astrocytes
did not express more Cx30 and Cx43 mRNA as well as
Our immunofluorescence analyses revealed, in accord-
ance with the unaltered level of Cx30, Cx36 and Cx43
protein, no change in location of these proteins within the
CA3 region and also other regions (i.e., dentate gyrus) of
kainate-treated and kindled rats (data not shown), when
compared to control sections. This is of some importance,
since Ochalski et al.  demonstrated that in rat thalamus
and striatum 3–6 days after direct kainate or NMDA
administration, hypertrophic astrocytes in the affected
areas responded with an intermediate internalization and
subsequent redistribution of Cx43 protein into their
filopodia. We found no hints for internalization or storage
of Cx43, Cx36 or Cx30 proteins. However, these simi-
larities in the immunofluorescence pattern in kainate-
treated or kindled rats, compared to controls, do not
exclude subtle pathological dysarrangements of gap junc-
tion distribution within astrocytic or neuronal clusters.
Even a small decrease of Cx43 and Cx30 in astrocytes may
disturb proper K -buffering or a reduction of Cx36
containing gap junctions between CA3 pyramidal neurons
or GABAergic interneurons could influence their capacity
to synchronize input stimuli. Subtle dysarrangements could
support the imbalance towards the long-term seizure prone
state of hippocampi in these epileptic rats, in analogy to
human epileptic tissue. With regard to Cx36, our results
with kainate-treated and kindled rats were slightly differ-
ent. In kainate-treated rats, some neuronal loss  might
result in the observed significant decrease of Cx36 mRNA,
whereas in kindled rats a significant transient decrease of
Cx36 mRNA and protein is more likely due to decreased
expression of Cx36 in hippocampal neurons. Interestingly,
Belluardo et al.  cloned and mapped the human Cx36
gene on chromosome 15q14, a locus which is linked to
JME (juvenile myoclonic epilepsy).
Lee et al.  investigated the permeability of gap
junction channels in cultured astrocytes from tissues
resected from medically intractable epilepsy patients. Gap
junction coupling was significantly increased only between
astrocytes cultured from epileptic foci that were associated
with cortical glial tumors or arose from the parahippocam-
pus. These results suggested that more subtle effects
concerning the permeability of gap junctions could in-
fluence the seizure-prone state in epileptic patients. In a
recent review  experimental data were summarized,
which described the fast modulation of gap junction
communication by intracellular pH and second messen-
gers. An increased electrotonic coupling seems to occur
during the K -mediated alkaline shift at the start of a
seizure-like event, followed by reduced coupling between
neurons caused by acidification at the end of a seizure .
Disturbances in this regulatory mechanism could also be
responsible for increased permeability, independent of the
amount of gap junction protein and gap junction channels.
Thus it is possible that local gap junction functions are
altered under conditions of epilepsy, even if the level of
connexin expression is not altered as we have found in two
rat models of human epilepsy.
This work was supported by grants of the Deutsche
Forschungsgemeinschaft through SFB 400 (B3) and the
Fonds der Chemischen Industrie to K.W., the SFB 507 to
U.H., and SFB 400 and the German-Israel Programme of
the BMBF to H.B.
 N. Belluardo, A. Trovato-Salinaro, G. Mudo, Y.L. Hurd, D.F.
Condorelli, Structure, chromosomal localization, and brain expres-
sion of human Cx36 gene, J. Neurosci. Res. 57 (1999) 740–752.
 E.C. Beyer, D.L. Paul, D.A. Goodenough, Connexin43: a protein
from rat heart homologous to a gap junction protein from liver, J.
Cell Biol. 105 (1997) 2621–2629.
 D.F. Condorelli, R. Parenti, F. Spinella, A. Trovato-Salinaro, N.
Belluardo, V. Cardile, F. Cicirata, Cloning of a new gap junction
gene (Cx36) highly expressed in mammalian brain neurons, Eur. J.
Neurosci. 10 (1998) 1202–1208.
 A.H. Cornell-Bell, S.M. Finkbeiner, M.S. Cooper, S.J. Smith,
Glutamate induced calcium waves in cultured astrocytes: Long-
range glial signaling, Science 247 (1990) 470–473.
 A. DaCunha, J.J. Jefferson, W.R. Tyor, J.D. Glass, F.S. Janotta, L.
Vitkovic, Gliosis in human brain: relationship to size but not other
properties of astrocytes, Brain Res. 600 (1993) 161–165.
 E. Dahl, D. Manthey, Y. Chen, H.J. Schwartz, Y.S. Chang, P.A.
Lalley, B.J. Nicholson, K. Willecke, Molecular cloning and func-
tional expression of mouse connexin-30, a gap junction gene highly
expressed in adult brain and skin, J. Biol. Chem. 271 (1996)
 J.W. Dani, A. Chernjavsky, S.J. Smith, Neuronal activity triggers
calcium waves in hippocampal astrocyte networks, Neuron 8 (1992)
 R. Dermietzel, Molecular diversity and plasticity of gap junctions in
the nervous system, in: D.C. Spray, R. Dermietzel (Eds.), Gap
Junctions in the Nervous System, Springer, New York, NY, 1996,
 R. Dermietzel, O. Traub, T.K. Hwang, E.C. Beyer, M.V.L. Bennett,
D.C. Spray, K. Willecke, Differential expression of three gap
junction proteins in developing and mature brain tissues, Proc. Natl.
Acad. Sci. USA 86 (1989) 10148–10151.
 A. Draguhn, R.D. Traub, D. Schmitz, J.G. Jefferys, Electrical
coupling underlies high-frequency oscillations in the hippocampus
in vitro, Nature 394 (1998) 189–192.
 K. Elisevich, S.A. Rempel, B.J. Smith, K. Edvardsen, Hippocampal
connexin43 expression in human complex partial seizure disorder,
Exp. Neurol. 145 (1997) 154–164.
 K. Elisevich, S.A. Rempel, B.J. Smith, N. Allar, Connexin 43
mRNA expression in two experimental models of epilepsy, Mol.
Chem. Neuropathol. 32 (1997) 75–88.
G. Sohl et al. / Molecular Brain Research 83 (2000) 44–51 51
 K. Elisevich, S.A. Rempel, B.J. Smith, K. Hirst, Temporal profile of
connexin43 mRNA expression in a tetanus toxin-induced seizure
disorder, Mol. Chem. Neuropathol. 35 (1998) 23–37.
 M.O.K. Enkvist, K.D. McCarthy, Activation of protein kinase C
blocks astroglial gap junction communication an inhibits the spread
of calcium waves, J. Neurochem. 59 (1992) 519–526.
 A. Hanauer, J.L. Mandel, The glyceraldehydephosphate dehydro-
genase gene family: structure of a human cDNA and of an X
chromosome linked pseudogene; amazing complexity of the gene
family in mouse, EMBO J. 3 (1984) 2627–2633.
 M. Khurgel, G.O. Ivy, Astrocytes in kindling: relevance to epi-
leptogenesis, Epilepsy Res. 26 (1996) 163–175.
 A. Konnerth, U. Heinemann, Y. Yaari, Nonsynaptic epileptogenesis
in the low mammalian hippocampus in vitro. Development of
seizure like activity in low extracellular calcium, J. Neurophysiol. 56
 P. Kunzelmann, W. Schroder, O. Traub, C. Steinhauser, R. Der-
mietzel, K. Willecke, Late onset and increasing expression of gap
junction protein connexin30 in adult murine brain and long-term
cultured astrocytes, Glia 25 (1999) 111–119.
 S.H. Lee, S. Magge, D.D. Spencer, H. Sontheimer, A.H. Cornell-
Bell, Human epileptic astrocytes exhibit increased gap junction
coupling, Glia 15 (1995) 195–202.
 S.K. Lewis, V. Krek, M. Shelanski, N.J. Cowan, Sequence of a
cDNA clone encoding mouse glial fibrillary acidic protein: structural
conservation of intermediate filaments, Proc. Natl. Acad. Sci. USA
81 (1984) 2743–2746.
 D. Manthey, F. Bukauskas, C.G. Lee, C. Kozak, K. Willecke,
Molecular cloning and functional expression of the mouse gap
junction gene connexin57 in human HeLa cells, J. Biol. Chem. 274
 J.I. Nagy, D. Patel, P.A.Y. Ochalski, G.L. Stelmack, Connexin30 in
rodent, cat and human brain: selective expression in gray matter
astrocytes, co-localisation with connexin43 at gap junctions and late
developmental appearance, Neuroscience 88 (1999) 447–468.
 C.C.G. Naus, J.F. Bechberger, D.L. Paul, Gap junction gene
expression in human seizure disorder, Exp. Neurol. 111 (1991)
 M. Nedergaard, Direct signaling from astrocytes to neurons in
cultures of mammalian brain cells, Science 263 (1994) 1768–1771.
 P.A. Ochalski, M.A. Sawchuk, E.L. Hertzberg, J.I. Nagy, Astrocytic
gap junction removal, connexin43 redistribution, and epitope mask-
ing at excitatory amino acid lesion sites in rat brain, Glia 14 (1995)
 D.L. Paul, Molecular cloning for the rat liver gap junction protein, J.
Cell Biol. 103 (1986) 123–134.
 G. Paxinos, C.Watson, in: The Rat Brain in Stereotaxic Coordinates,
2nd Edition, Academic Press, Orlando, FL, 1986.
 J.L. Perez Velazquez, P.L. Carlen, Gap Junctions, synchrony and
seizures, Trends Neurosci. 23 (2000) 68–74.
 R.J. Racine, Modification of seizure activity by electrical stimula-
tion: II. Motor seizure, Electroencephalogr. clin. Neurophysiol. 32
 J. Sambrook, E.F. Fritsch, T. Maniatis, in: A Laboratory Manual,
2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor,
 J.S. Schweitzer, P.R. Patrylo, F.E. Dudek, Prolonged field bursts in
the dentate gyrus: dependence in low calcium, high potassium and
nonsynaptic mechanisms, J. Neurophysiol. 68 (1992) 2016–2025.
 A.M. Simon, D.A. Goodenough, Diverse function of vertebrate gap
junctions, Trends Cell. Biol. 12 (1998) 477–483.
 G. Sohl, J. Degen, B. Teubner, K. Willecke, The murine gap
junction gene connexin36 is highly expressed in the mouse retina
and regulated during brain development, FEBS Lett. 428 (1998)
 G. Sperk, H. Lassmann, H. Baran, S.J. Kish, F. Seitelberger, O.
Hornykiewicz, Kainic acid induced seizures: neurochemical and
histopathological changes, Neuroscience 10 (1983) 1301–1315.
 M. Srinivas, R. Rozental, T. Kojima, R. Dermietzel, M. Mehler,
D.F. Condorelli, J.A. Kessler, D.C. Spray, Functional properties of
channels formed by the neuronal gap junction protein connexin36, J.
Neurosci. 19 (1999) 9848–9855.
 O. Steward, E.R. Torre, R. Tomasulo, E. Lothmann, Seizures and
the regulation of astroglial gene expression, Epilepsy Res. Suppl. 7.
(Chapter 14) (1992) 197 pp.
 B. Teubner, J. Degen, G. Sohl, M. Guldenagel, F.F. Bukauskas, E.B.
Trexler, V.K. Verselis, C.I. De Zeeuw, C.G. Lee, C.A. Kozak, R.
Dermietzel, K. Willecke, Functional expression of the murine
connexin 36 gene coding for a neuron specific gap junctional
protein, J. Mem. Biol. 176 (2000) 249–262.
 O. Traub, R. Eckert, H. Lichtenberg-Frate, C. Elfgang, B. Bastide,
K.H. Scheidtmann, D.F. Hulser, K. Willecke, Immunochemical and
electrophysiological characterisation of murine connexin40 and -43
in mouse tissue and transfected human cells, Eur. J. Cell Biol. 64
 O. Traub, J. Look, R. Dermietzel, F. Brummer, D. Hulser, K.
Willecke, Comparative characterisation of the 21-kDa and 26-kDa
gap junction protein in the murine liver and cultured hepatocytes,
Eur. J. Cell Biol. 108 (1989) 1039–1051.
 R.D. Traub, D. Schmitz, J.G. Jefferys, A. Draguhn, High-frequency
population oscillations are predicted to occur in hippocampal
pyramidal neuronal networks interconnected by axoaxonal gap
junctions, Neuroscience 92 (1999) 407–426.
 T.A. Valiante, J.L. Perez-Velazquez, S.S. Jahromi, P.L. Carlen,
Coupling potentials in CA1 neurons during calcium-free induced
field burst activity, J. Neurol. 10 (1995) 405–410.
 J.Veliskova, L.Velisek, P. Mares, Epileptic phenomena produced by
kainic acid in laboratory rats during ontogenesis, Physiol. Bohem.
37 (5) (1988) 395–405.
 C. Venance, D. Piomelli, J. Glowinski, C. Giaume, Inhibition by
anandamide of gap junction and intercellular calcium signaling in
striatal astrocytes, J. Neurosci. 15 (1995) 6946–6956.
 Z.Q. Xiong, P. Saggau, J.L. Stringer, Activity-dependent intracellu-
lar acidification correlates with the duration of seizure activity, J.
Neurosci. 20 (2000) 1290–1296.
 T. Yamamoto, A. Ochalski, E.L. Hertzberg, J.I. Nagy, On the
organisation of astrocytic gap junctions in rat brain as suggested by
LM and EM immunohistochemistry of connexin43 expression, J.
Comp. Neurol. 302 (1990) 853–883.