Downregulation of Potassium Chloride Cotransporter KCC2 After Transient Focal Cerebral Ischemia
Background and Purpose— The potassium chloride cotransporter 2 (KCC2) is the main neuronal chloride extruder in the adult nervous system. Therefore, KCC2 is responsible for an inwardly directed electrochemical gradient of chloride that leads to hyperpolarizing GABA-mediated responses. Under some pathophysiological conditions, GABA has been reported to be depolarizing because of a downregulation of KCC2. This is the first study to our knowledge analyzing the expression of KCC2 after a focal cerebral ischemia.
Methods— Mild and severe ischemia were induced in rats by a transient occlusion of the middle cerebral artery for 30 and 120 minutes, respectively. KCC2 mRNA and protein expression were studied in the ischemic hemisphere after different reperfusion times (2 hour, 1 day, 7 days, 30 days, 168 days) by using quantitative polymerase chain reaction, Western blotting, and immunohistological staining.
Results— We found a substantial decrease of KCC2 mRNA and protein levels in the ischemic hemisphere, with a stronger downregulation of KCC2 after severe vs mild ischemia. Long-term surviving cells expressing KCC2 could be detected in the infarct core. These cells were identified as GABAergic interneurons mainly expressing parvalbumin.
Conclusions— Our study revealed a substantial neuron-specific downregulation of KCC2 after focal cerebral ischemia.
Cation chloride cotransporters play an essential role in the regulation of neuronal chloride homeostasis. The neuron-specific isoform potassium chloride cotransporter 2 (KCC2) is the main chloride extruder in the adult nervous system.1,2⇓ Various mechanisms by which KCC2 activity is regulated are a main focus of current research.3,4⇓ KCC2 is responsible for an inwardly directed electrochemical gradient for chloride that leads to hyperpolarizing GABAA receptor-mediated responses in the adult brain.2,5⇓ Soon after birth, KCC2 expression, especially in the hippocampus and the cortex, is low but developmentally upregulated to adult levels during the first 2 postnatal weeks.2,6–9⇓⇓⇓⇓ A transient excitatory action of GABA in the immature brain might be important for the development and maturation of the nervous system.10 Under certain pathophysiological conditions, eg, neuronal trauma,11 axotomy of motoneurons,12,13⇓ metabolic inhibition,14 and epilepsy,15–17⇓⇓ GABA has also been reported to be depolarizing or even excitatory. A downregulation of KCC2 could be one reason for a depolarizing or even an excitatory GABA action. A decrease of KCC2 expression was described in the aforementioned studies12,13,15–17⇓⇓⇓⇓ and further after pilocarpine-induced epilepsy,18 as well as in cortical malformations associated with epilepsy.19 A downregulation of KCC2 expression was also revealed under ischemic conditions, particularly after oxygen glucose deprivation in hippocampal slices20 and in an in vivo model of global ischemia.21 There are no studies addressing changes of KCC2 expression after a focal cerebral infarction. Focal cerebral ischemia results in typical pathophysiological events with a spatiotemporal dynamic. The infarct core is mainly characterized by necrotic cell damage, whereas perilesional regions surrounding the infarct core are severely but not irreversibly impaired in their physiological processes.22 Previous studies have demonstrated reorganization processes and plasticity in the brain after a focal ischemic insult.23 Neurons in perilesional regions can acquire the function of irreversibly damaged cells; dendrites and axons grow out, arborize, and new synaptic connections are generated.24,25⇓ Further research showed survival of GABAergic interneurons after ischemia in perilesional regions and even in directly affected areas.26,27⇓ On the contrary, delayed neuronal death in regions surrounding the infarct is also a consequence of cerebral ischemia. As already mentioned, KCC2 is necessary for the hyperpolarizing effect of GABA in the adult brain. Therefore, an attenuated postischemic neuronal KCC2 expression may initiate depolarizing GABA responses, thereby influencing plasticity and secondary damage after stroke. In the present study, we analyzed the expression of KCC2 after a transient middle cerebral artery occlusion (MCAO) for 30 minutes and 120 minutes leading to mild and severe injury, respectively. Postischemic expression of KCC2 was analyzed on transcript and protein levels, and immunohistochemical staining of KCC2 was performed at various reperfusion times (2 hours, 1 day, 7 days, 30 days, and 168 days).
Materials and Methods
Male Wistar rats aged 2 to 3 months, weighing 270 to 300 grams, were used. All animal procedures were approved by the local government (Thueringer Landesamt, Weimar) and conformed to international guidelines on the ethical use of animals. Animals were obtained from the animal facility of our institute (SEK, Service Einheit Kleinnager, Jena). Efforts were made to reduce the number of animals used and their suffering. All surgeries were performed under deep anesthesia (isoflurane).
Induction of Focal Cerebral Ischemia by MCAO
Transient MCAO was induced according to Nagasawa and Kogure28 and modified by us, as recently described in detail.29 In brief, rats were anesthetized with isoflurane and a commercial 4-0 monofilament nylon suture coated with silicone rubber on the tip (0.35±0.02 mm diameter; 35SPRe, Doccol Corp) was introduced through an incision of the right common carotid artery into the internal carotid artery to occlude the origin of the right middle cerebral artery (MCA). After 30 minutes or 120 minutes of occlusion, the suture was withdrawn. Sham-operated animals underwent the same procedure without occlusion of the MCA.
For quantitative polymerase chain reaction and Western blotting (2 hours, 1 day, 7 days, and 30 days after reperfusion), brains were removed after cervical dislocation. Using a rat brain slicer (rodent brain matrix, adult rat, coronal sections; ASI Instruments), 3-mm coronal sections were dissected according to Popp et al.29 Coronal sections comprising the infarct (bregma, 1.0 to −2.0 mm, ±0.5 mm)30 were separated into ipsilateral and contralateral hemispheres, snap-frozen, and stored at −80°C until use. Adjacent sections were used for infarct validation by TTC staining (2% TTC in 0.9% NaCl at 37°C for 10 minutes). For immunohistochemistry (1 day, 7 days, 30 days, and 168 days after reperfusion), deeply anesthetized rats were fixed by perfusion through the ascending aorta with 4% paraformaldehyde. Brains were removed, cryoprotected in 0.2 mol/L phosphate-buffered saline containing 30% sucrose, and stored at −80°C. Coronal sections were cut at 30 μm on a freezing microtome (Microm International GmbH, Thermo Scientific).
Quantitative Polymerase Chain Reaction
For quantitative polymerase chain reaction, total RNA (30 minutes of MCAO, n=24; sham, n=20); (120 minutes of MCAO, n=12; sham, n=10) was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen). Quantitative polymerase chain reaction was performed as described previously.31 Specific primers for KCC2 (forward, AGG TGG AAG TCG TGG AGA TG; reverse, CGA GTG TTG GCT GGA TTC TT) were designed by the use of Primer3 design software based on the published sequence for KCC2 (NM_134363). Quantitative polymerase chain reaction was performed in 20 μL amplification mixture consisting of RT2 Real-Time SYBR green polymerase chain reaction master mix (SABiosciences Corporation), cDNA (equivalent to 25 ng reverse-transcribed RNA) and primers (250 nM final concentration each). Specific transcripts were amplified with Rotor Gene 6000 (LTF-Labortechnik/Corbett Life Science). GAPDH (NM_017008) was used as reference gene (primer forward, GCA TTG CTC TCA ATG ACA ACT T; primer reverse, GGC CTC TCT CTT GCT CTC AGT). KCC2 ratios (MCAO vs sham) were calculated using the Pfaffl equation.32
Western Blot Analysis
For Western blotting coronal slices of ipsilateral and contralateral hemispheres (30 minutes of MCAO, n=15; sham, n=12; 120 minutes of MCAO, n=4; sham, n=3) were homogenized (Miccra-D1; Art) in 10 volumes of ice-cold homogenization buffer (0.32 mol/L sucrose; 4 mmol/L Tris–HCl, pH 7.4; 1 mmol/L EDTA; and 0.25 mmol/L dithiothreitol) containing protease inhibitors (Complete Mini; Roche). After ultrasonic treatment and a subsequent incubation for 10 minutes at 4°C the homogenates were centrifuged at 1000×g for 15 minutes at 4°C. The supernatant containing whole protein was taken, stored at −80°C, and used for further analyses. Equal amounts of protein (5 μg or 10 μg) obtained from sections of ipsilateral or contralateral hemisphere were pooled (n=3) or individually evaluated (30 minutes of MCAO, n=6; 120 minutes of MCAO, n=4). Samples were diluted in 5× SDS buffer, denaturated at 50°C or 95°C for 15 minutes, and separated onto an 8% SDS-polyacrylamide gel. Proteins were transferred by electro blotting (BioRad) onto a polyvinylidene fluoride membrane (Hybond P; GE Healthcare). Membranes were incubated with primary antibodies raised against KCC2 (07-432, 1:2000; Upstate), NeuN (MAB377, 1:2000; Chemicon) or β-actin (AB8227, 1:30000; Abcam) in 5% blocking solution (5% nonfat milk powder, 0,1% Tween20 in 1× phosphate-buffered saline) for 18 hours at 4°C, followed by incubation with horseradish peroxidase-conjugated goat antimouse IgG antibody (1:5000, sc-2002) or goat antirabbit IgG antibody (1:20 000, sc-2004; both from Santa Cruz Biotechnology). Proteins were detected by enhanced chemiluminescence (ECL Plus Western Blot Detection System; GE Healthcare) and analyzed with the image acquisition system LAS 3000 (Fujifilm). Optical density of protein bands was quantified with the AIDA Image Analyzer Software (Raytest).
Immunohistochemistry (KCC2, NeuN, MAP2, GAD67, Parvalbumin)
Free-floating sections (30 minutes of MCAO, n=12; 120 minutes of MCAO, n=3) were treated with 0.24% H2O2 before the antibodies were applied in Tris-buffered saline containing 3% normal donkey serum and 0.2% Triton X-100. Sections were incubated at 4°C overnight with antibody against either KCC2 (07-432, 1:2000; Upstate), GAD67 (goat anti-GAD67, 1:5000; Chemicon), or parvalbumin (mouse anti-parvalbumin, 1:1000; Swant) and further processed by the Vectastain Elite ABC Kit (Vector Laboratories) using a donkey anti-mouse or donkey anti-rabbit biotinylated secondary antibody (Dianova, Germany). Immunoreactivity was developed in 3.3′-diaminobenzidine tetrahydrochloride (DAB; Sigma). For colabeling studies the primary antibody of KCC2 was simultaneously applied with either NeuN (mouse anti-NeuN, 1:500; Chemicon), MAP2 (mouse anti-MAP2ab, 1:250; Sigma) GAD67, or parvalbumin antibody and were detected by fluorescence-labeled secondary antibodies (Alexa488 donkey anti-goat, Alexa488 donkey anti-mouse, and Rhodamine donkey anti-rabbit; Molecular Probes Inc, Invitrogen). Slices were analyzed via confocal microscopy (LSM 510; Carl Zeiss MicroImaging GmbH).
Statistical significance was determined using Mann–Whitney U test (P≤0.05; P≤0.01). Statistical analyses were performed using SPSS (SPSS GmbH Software).
Infarct Validation: Mild vs Severe Ischemia
The infarct of all animals included in this study was validated by TTC staining (bregma, 4.0 to 1.0 mm, ±0.5 mm). After a mild 30-minute MCAO, the infarct core was mainly restricted to the striatum (caudate putamen). By contrast, after 120-minute MCAO the infarct core was enlarged and always comprised cortical areas (Figure 1).
Downregulation of KCC2 mRNA and Protein
After 30-minute MCAO, KCC2 mRNA expression, normalized to GAPDH, was found to be unaltered 2 hours after reperfusion in the ipsilateral hemisphere (bregma, 1.0 to −2.0 mm, ±0.5 mm). At 1 day after reperfusion, a significant decrease in KCC2 mRNA of ≈30% could be detected in the ischemic hemisphere, and this downregulation further decreased to ≈50% until 7 days (P≤0.05; Figure 2A). KCC2 expression at 30 days of reperfusion was found to be unaltered. As a consequence of severe ischemia (120 minutes of MCAO), KCC2 mRNA was more strongly downregulated to ≈50% already at 1 day of reperfusion and to ≈40% at 7 days of reperfusion (P≤0.01; Figure 2B). Analysis of sham-operated animals revealed a constant KCC2 expression in the ipsilateral compared to the contralateral hemisphere normalized to GAPDH (data not shown).
A downregulation of KCC2 protein expression, normalized to β-actin, was found in the ischemic hemisphere starting at 2 hours of reperfusion after mild ischemia (pool of 3 rats; Figure 3A). At 30 days of reperfusion, individual samples were processed (n=6), thereby revealing a significant downregulation of KCC2 protein expression (to ≈42%; P≤0.05; Figure 3B). After severe ischemia protein expression of KCC2 was analyzed at 7 days of reperfusion on individual samples (n=4), thereby indicating a drastic downregulation of KCC2 protein expression (to ≈17%) in the ischemic hemisphere (Figure 3C). In the contralateral hemisphere, the expression of KCC2 protein was found to be unaltered after mild and severe ischemia (Figure 3D–F).
Because KCC2 is expressed specifically in neurons, we additionally normalized the protein data to NeuN as a neuronal reference protein. Western blotting revealed a reperfusion time-dependent decrease in NeuN expression after ischemia in the ipsilateral hemisphere (Figure 4A). In particular, neuronal cell death after 30 minutes of MCAO was found to be significant (decrease of NeuN to ≈70% at 30 days of reperfusion compared to sham P≤0.05; Figure 4B). A drastic downregulation of NeuN protein expression (to ≈27%) was found in the ischemic hemisphere after severe ischemia at a time when β-actin expression was unaltered (Figure 4C). In the contralateral hemisphere, no significant alterations of both β-actin and NeuN protein expression were detected after mild as well as severe ischemia (Figure 4D–F).
KCC2 protein expression in the ischemic hemisphere, normalized to NeuN, was found to be downregulated starting at 1 day of reperfusion but to a lesser extent compared to the β-actin normalization (Figures 5A, 3⇑A). At 30 days of reperfusion individual samples were processed (n=6), thereby revealing a significant decrease of KCC2 protein (to ≈66%) (P≤0.05; Figure 5B). After severe ischemia, KCC2 protein expression was found to be downregulated (to ≈56%) in the ischemic hemisphere again, to a lesser extent than normalized to β-actin (Figures 5C, 3⇑C). Contralateral KCC2 expression was unaltered after a mild ischemia. After severe ischemia, a trend of KCC2 downregulation (to ≈66%) was shown that is not significant (Figure 5F).
KCC2-Positive Cells and GABAergic Interneurons in the Striatal Ischemic Core 1 Day, 7 Days, and 168 Days After 30 Minutes of MCAO
In the contralateral hemisphere and in sham-operated rats, KCC2 showed a diffuse neuropil staining and an intense staining outlining the somata and proximal dendrites of neurons. The infarct core was characterized by a reduced expression of KCC2. Regions adjacent to the infarct core seem to be stained less intensely but no deviation in the histological pattern of KCC2 expression could be observed (Figure 6A). Cells with an intense labeling of KCC2 in their neuronal cell membrane and in their dendrites were detected in the striatal ischemic core at least up to 168 days (Figure 6B, C). Dendrites of KCC2-positive cells were colocalized with the cytoskeletal marker MAP2 (Figure 6E), thereby presenting the characteristic beading as already described previously under ischemic conditions.33 MCAO for 120 minutes did not reveal KCC2-positive neurons in the infarct core because of the extended necrotic processes, but a similar phenomenon was observed in perilesional regions (data not shown). Qualitative analyses of KCC2 positive cells at 7 days after ischemia showed that KCC2-positive cells in the infarct core were colabeled with GAD67 (89.33%±8.33%; Figure 6F) and with parvalbumin (73.33%±8.33%; Figure 6G). A relatively small number of KCC2-positive cells also expressed calretinin (17.33%±6.11%). Therefore, the majority of KCC2-positive cells surviving a mild ischemia were identified as striatal GABAergic interneurons containing parvalbumin. Somatostatin-positive interneurons and cholinergic interneurons did not express KCC2 (data not shown).
Immunohistological staining further revealed a minor loss of striatal GABAergic interneurons in the ischemic infarct core when compared to analogous contralateral regions (ratio of surviving cells: GAD67, 67.08%±13.03%; parvalbumin, 77.51%±10.41%; Figure 6H–L). GAD67 was weakly stained in the contralateral striatum and showed an increase of signal intensity in the striatal infarct core, whereas parvalbumin was diminished in ischemia surviving cells (Figure 6I–L). An increase of GAD staining intensity (probably attributable to an enhanced metabolic activity) has also been observed previously under epileptic and ischemic conditions.34–36⇓⇓ The decrease of parvalbumin in GABAergic interneurons after ischemia is in agreement with a previous study.37
A Substantial Downregulation of KCC2 After Stroke
Here, we revealed for the first time to our knowledge a sustained postischemic downregulation of KCC2 after focal cerebral ischemia of different degree of severity. The KCC2 decrease initially detected on mRNA level at 1 day and at 7 days after reperfusion could be confirmed on protein level. At 30 days after mild ischemia, RNA regulation was not found to be accompanied by protein synthesis in the same way. KCC2 mRNA expression recovered, whereas the protein level was still significantly decreased at this time. There might be various reasons for that discrepancy, here restricted to the late reperfusion time, eg, the transcription efficacy or various posttranscriptional mechanisms controlling the mRNA lifetime and the protein translation rate. Therefore, the determination of mRNA level is often only a first indicator for the functionally more important protein level. At the protein level, a long-lasting and profound downregulation of KCC2 was revealed up to 30 days after a mild ischemia and up to 7 days after severe ischemia. A longer time of reperfusion could not be realized after severe ischemia because of an excessive mortality rate.
Functional Implications of Postischemic Downregulation of KCC2
A decrease of KCC2 expression was previously described after certain pathologies.12,13,15–19,38⇓⇓⇓⇓⇓⇓⇓ An attenuated KCC2 expression was also found under ischemic conditions.20 Galeffi et al20 specified the downregulation of KCC2 protein expression after oxygen-glucose deprivation as one possible reason for an increase in intracellular chloride, an effect that was found after different models of cerebral ischemia.39 An increase in intracellular chloride can cause depolarizing or even excitatory GABAA responses.12–14⇓⇓ Determination of the intracellular chloride level in neurons after focal cerebral ischemia would be of great interest for the functional significance of the here found KCC2 downregulation. The fact that we have not pinpointed the downregulation of KCC2 expression to a specific cell type yet makes this experimental approach hardly feasible. In addition, electrophysiological determination of the chloride equilibrium potential by using perforated patch-clamp recordings is complicated by the fact that we had to investigate adult tissue with an ischemic damage. A further possible approach to clarify the functional relevance of KCC2 would be the use of knockout mice. Unfortunately, KCC2 knockout mice die within the 2 postnatal weeks because of severe motor deficits or because of spontaneous generalized seizures.5,46⇓ A conditional knockout mouse of KCC2 is not available yet. In addition, there is no specific pharmacological tool available to manipulate KCC2 function in vivo.
We hypothesize that the downregulation of KCC2 would lead to a positive shift of the chloride equilibrium potential, which is expected to reduce functional GABAergic inhibition. It is important to note that this not necessarily requires a depolarizing effect of GABAergic synaptic transmission. Even a reduction in the hyperpolarizing action of GABA might promote potential mechanisms of plasticity and recovery. Previous studies have demonstrated reorganization processes and plasticity in the brain after a focal ischemic insult.23 Postischemic plasticity displays some developmental features.40 In the immature brain, depolarizing GABA is important for the development of the nervous system.41,42⇓ The downregulation of KCC2 after stroke, analogous to the development, therefore might initiate reorganization of neuronal networks, eg, by axonal outgrowth, newly formed (or uncovering of silent preexisting) synapses, and establishing of new contacts.24,43⇓
However, a decrease in KCC2 expression may turn out to be negative in the case when the shift of the chloride equilibrium potential leads to GABA-mediated depolarization. Depolarizing GABA might activate postischemic secondary cascades that finally initiate delayed neuronal cell death. Membrane depolarization leads to an increase in intracellular calcium concentration via activation of voltage-gated calcium channels.12,13⇓ In addition, depolarizing GABA might relieve the Mg2+ block of NMDA receptors, thereby leading to a further calcium increase. This might result in a cytosolic calcium overload, especially when a sustained excitatory input can no longer be compensated.44 An adverse impact of GABA-mediated depolarization during ischemic episodes could also be revealed by Ben-Ari et al.45 In a very elegant study they could show that neurons of newborns were protected from a hypoxic insult occurring during delivery by oxytocin-mediated excitatory-to-inhibitory switch of GABA actions via a transient reduction of intracellular chloride.45
Another interesting aspect is that postischemic KCC2 downregulation might be involved in the pathogenesis of epilepsy after stroke. Epileptic seizures occur in up to 25% of stroke victims.23 Investigations after an epileptogenic injury in rodents17 and in human epileptic tissues15,16⇓ revealed an attenuated KCC2 expression associated with an impaired GABA response. Although it is not completely clear if this downregulation of KCC2 is a consequence or the cause of epilepsy, an altered regulation of chloride transporters is considered to contribute to the hyperexcitability finally leading to epileptic seizures.16,17,19⇓⇓
NeuN Rather Than β-Actin Is More Suitable for Normalization of Proteins Specifically Expressed in Neurons
β-Actin is a cytoskeletal structure protein ubiquitously expressed in all cell types and widely accepted as a reference protein. In the infarct core, neurons and astrocytes underlie irreversible cell death. Within a few days a glial scar is formed surrounding the infarct, mainly consisting of proliferative astrocytes.47 Neurons in perilesional areas are still vulnerable to apoptotic ischemic death. These processes—proliferation of astrocytes in some cases vs delayed neuronal death in other cases—result in a shift in cell populations. Because β-actin is ubiquitously expressed, a shift in cell type proportion cannot be detected by using this reference protein; furthermore, a cell type-specific regulation of the protein of interest cannot be revealed.
Our Western blot study comparing β-actin and NeuN indicated that β-actin might not always be recommended as an optimal reference protein after stroke. After normalization of KCC2 expression data to NeuN, a significant postischemic downregulation of KCC2 protein was revealed, specifically in the neuronal cell population. This effect was more pronounced after severe (vs mild) ischemia. Nevertheless, our results indicate that the KCC2 decrease after an ischemic insult represents not the consequence of simple cells loss but rather an active mechanism of the surviving neurons to downregulate their KCC2 level.
KCC2-Positive Cells Found in the Striatal Infarct Core Were Identified as GABAergic Interneurons
By immunhistological studies, a long-term survival (up to 6 months) of KCC2-positive cells was found in the striatal infarct core after a mild focal ischemia. Mild ischemia is known to preserve the majority of striatal interneurons against neuronal cell death in mouse.26 This is in agreement with our study in which ≈80% of GABAergic interneurons in the striatum survived a mild transient focal ischemia in rats. Here, we found a long-term survival (up to 6 months) of KCC2-positive cells in the infarct core after a mild focal ischemia. Surviving KCC2-positive cells were identified as striatal GABAergic interneurons (GAD67) mainly colocalized with parvalbumin and to a lesser extent with calretinin. A sustained expression of KCC2 in surviving parvalbumin-containing interneurons, analogous to our findings in the striatum, was also revealed in the hippocampus after 4-vessel occlusion.21 Parvalbumin and calretinin are calcium-binding proteins, and thus these subtypes of GABAergic interneurons are less vulnerable to mild ischemic stress because of their capability to buffer cytotoxic levels of intracellular calcium. Therefore, GABAergic interneurons might secure themselves against excitotoxicity through calcium buffering and KCC2-mediated chloride extrusion. Although these neurons can be detected in the long-term after stroke, we do not know if they are connected to other neurons or if they have any functional relevance in reorganization processes of perilesional areas.
The authors thank Svetlana Tausch, Diana Freitag, and Madlen Guenther for their excellent technical assistance. The authors are grateful to Christian Huebner, Knut Holthoff, and Knut Kirmse for their insightful comments.
Sources of Funding
This work was supported by Bundesministerium für Bildung und Forschung 01GZ0709.
- Received October 13, 2009.
- Revision received November 2, 2009.
- Accepted November 4, 2009.
- ↵Payne JA, Stevenson TJ, Donaldson LF. Molecular characterization of a putative k-cl cotransporter in rat brain. A neuronal-specific isoform. J Biol Chem. 1996; 271: 16245–16252.
- ↵Balakrishnan V, Becker M, Lo?hrke S, Nothwang HG, Guresir E, Friauf E. Expression and function of chloride transporters during development of inhibitory neurotransmission in the auditory brainstem. J Neurosci. 2003; 23: 4134–4145.
- ↵Ben-Ari Y, Gaiarsa J, Tyzio R, Khazipov R. Gaba: A pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev. 2007; 87: 1215.
- ↵van den Pol A, Obrietan K, Chen G. Excitatory actions of gaba after neuronal trauma. J Neurosci. 1996; 16: 4283.
- ↵Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, Akaike N. Reduction of kcc2 expression and gabaa receptor-mediated excitation after in vivo axonal injury. J Neurosci. 2002; 22: 4412.
- ↵Toyoda H, Ohno K, Yamada J, Ikeda M, Okabe A, Sato K, Hashimoto K, Fukuda A. Induction of nmda and gabaa receptor-mediated ca2+ oscillations with kcc2 mrna downregulation in injured facial motoneurons. J Neurophysiol. 2003; 89: 1353–1362.
- ↵Huberfeld G, Wittner L, Clemenceau S, Baulac M, Kaila K, Miles R, Rivera C. Perturbed chloride homeostasis and gabaergic signaling in human temporal lobe epilepsy. J Neurosci. 2007; 27: 9866–9873.
- ↵Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, Ragozzino D, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F. Anomalous levels of cl- transporters in the hippocampal subiculum from temporal lobe epilepsy patients make gaba excitatory. Proc Natl Acad Sci U S A. 2006; 103: 8465–8468.
- ↵Pathak HR, Weissinger F, Terunuma M, Carlson GC, Hsu F-C, Moss SJ, Coulter DA. Disrupted dentate granule cell chloride regulation enhances synaptic excitability during development of temporal lobe epilepsy. J Neurosci. 2007; 27: 14012–14022.
- ↵Aronica E, Boer K, Redeker S, Spliet WGM, van Rijen PC, Troost D, Gorter JA. Differential expression patterns of chloride transporters, na+-k+-2cl–cotransporter and k+-cl–cotransporter, in epilepsy-associated malformations of cortical development. Neuroscience. 2007; 145: 185–196.
- ↵Galeffi F, Sah R, Pond BB, George A, Schwartz-Bloom RD. Changes in intracellular chloride after oxygen-glucose deprivation of the adult hippocampal slice: Effect of diazepam. J Neurosci. 2004; 24: 4478–4488.
- ↵Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke. 1989; 20: 1037–1043.
- ↵Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Sydney, Australia: Academic Press; 2005.
- ↵Sieber MW, Guenther M, Kohl M, Witte OW, Claus RA, Frahm C Inter-age variability of bona fide unvaried transcripts normalization of quantitative PCR data in ischemic stroke. Neurobiol Aging. 2008. In press.
- ↵Pfaffl MW. A new mathematical model for relative quantification in real-time rt-pcr. Nucleic Acids Res. 2001; 29: e45.
- ↵Li P, Murphy TH. Two-photon imaging during prolonged middle cerebral artery occlusion in mice reveals recovery of dendritic structure after reperfusion. J Neurosci. 2008; 28: 11970–11979.
- ↵Knopp A, Frahm C, Fidzinski P, Witte OW, Behr J. Loss of gabaergic neurons in the subiculum and its functional implications in temporal lobe epilepsy. Brain. 2008; 131: 1516–1527.
- ↵Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, Nanobashvili A, Kokaia Z, Airaksinen MS, Voipio J, Kaila K, Saarma M. Bdnf-induced trkb activation down-regulates the k+-cl- cotransporter kcc2 and impairs neuronal cl- extrusion. J Cell Biol. 2002; 159: 747–752.
- ↵Kristian T, Siesjo BK. Calcium in ischemic cell death. Stroke. 1998; 29: 705–718.
- ↵Tyzio R, Cossart R, Khalilov I, Minlebaev M, Hubner CA, Represa A, Ben-Ari Y, Khazipov R. Maternal oxytocin triggers a transient inhibitory switch in gaba signaling in the fetal brain during delivery. Science. 2006; 314: 1788–1792.