Cation-Chloride Cotransporters and GABA-ergic Innervation in the Human Epileptic Hippocampus

Epilepsia, 48(4):663–673, 2007Blackwell Publishing, Inc.C© 2007 International League Against Epilepsy

Cation-Chloride Cotransporters and GABA-ergic Innervationin the Human Epileptic Hippocampus

∗†Alberto Munoz, ∗Pablo Mendez, ∗Javier DeFelipe, and ‡Francisco Javier Alvarez-Leefmans

∗Instituto Cajal, CSIC, Madrid, Spain; †Department of Cell Biology, Universidad Complutense, Madrid, Spain;and ‡Department of Pharmacology and Toxicology, Wright State University, Dayton, Ohio, U.S.A.

Summary: Intracellular chloride concentration, [Cl−]i, deter-mines the polarity of GABAA-induced neuronal Cl− currents.In neurons, [Cl−]i is set by the activity of Na+, K+, 2Cl− co-transporters (NKCC) such as NKCC1, which physiologicallyaccumulate Cl− in the cell, and Cl− extruding K+, Cl− cotrans-porters like KCC2. Alterations in the balance of NKCC1 andKCC2 activity may determine the switch from hyperpolarizingto depolarizing effects of GABA, reported in the subiculum ofepileptic patients with hippocampal sclerosis. We studied theexpression of NKCC (putative NKCC1) and KCC2 in humannormal temporal neocortex by Western blot analysis and in nor-mal and epileptic regions of the subiculum and the hippocampusproper using immunocytochemistry. Western blot analysis re-vealed NKCC and KCC2 proteins in adult human neocorticalmembranes similar to those in rat neocortex.

NKCC and KCC2 immunolabeling of pyramidal and non-pyramidal cells was found in normal and epileptic hippocam-

pal formation. In the transition between the subiculum withsclerotic regions of CA1, known to exhibit epileptogenic ac-tivity, double immunolabeling of NKCC and KCC2 revealedthat approximately 20% of the NKCC-immunoreactive neu-rons do not express KCC2. In these same areas some neuronswere distinctly hyperinnervated by parvalbumin (PV) positivehypertrophic basket formations that innervated mostly neu-rons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket forma-tions also innervated KCC2-positive (76%) and -negative (24%)neurons. The data suggest that changes in the relative ex-pression of NKCC1 and KCC2 in neurons having aberrantGABA-ergic hyperinnervation may contribute to epileptiformactivity in the subicular regions adjacent to sclerotic areasof the hippocampus. Key Words: Epilepsy—Subiculum—Hippocampal sclerosis—Inhibition—NKCC1—KCC2.

Hippocampal sclerosis, a common feature of patientswith temporal lobe epilepsy, is characterized by cell lossand gliosis in various regions of the hippocampal for-mation, notably CA1, leaving the subiculum mostly in-tact (Honavar and Meldrum, 1997). Electrophysiologicalrecordings from human temporal lobe slices from epilep-tic patients with hippocampal sclerosis revealed sponta-neous synchronous interictal-like discharges, similar tothose recorded in intracranial electroencephalograms fromthe same patients (Cohen et al., 2002). The interictal-like discharges were initiated in the subiculum and in itstransitional area with CA1. The subicular circuit display-ing these interictal-like discharges includes interneuronsand a subgroup of pyramidal cells that are depolarizedby GABA released from interneurons. This depolarizingGABA-ergic signaling is likely to be involved in the gener-ation of these spontaneous interictal discharges in the tem-poral lobe of epileptic patients with hippocampal sclerosis

Accepted October 20, 2006.Address correspondence and reprint requests to A. Munoz, Instituto

Cajal, Av. Dr. Arce 37, 28002 Madrid. E-mail: [email protected]: 10.1111/j.1528-1167.2007.00986.x

(Cohen et al., 2002; Deisz, 2002). The direction and mag-nitude of GABA-induced Cl− currents through GABAA

receptors is determined by the intracellular [Cl−] result-ing mostly from the functional expression of the Na+-K+-2Cl− cotransporters (NKCCs) that accumulate Cl− insidethe cells, and the Cl− extruding K+-Cl− cotransporters(KCCs) (Alvarez-Leefmans, 1990; Alvarez-Leefmanset al., 2001; Delpire and Mount, 2002; Gamba, 2005).Alterations in the balance of NKCCs and KCCs activ-ity may determine the switch from a hyperpolarizing toa depolarizing effect of GABA, thereby contributing toepileptogenesis in human hippocampal formation (Cohenet al., 2003; Fukuda, 2005; Dzhala et al., 2005).

Various lines of evidence correlate epileptogenesis withaltered functional expression of NKCC and KCC trans-porters. Accordingly, deletion of KCC2 gene expressionin mice causes hyperexcitability in the hippocampus, gen-eralized seizures, and death shortly after birth (Woo et al.,2002). These changes are accompanied by loss of neuronspositive for parvalbumin (PV), which are presumed to beinhibitory GABA-ergic interneurons. In human temporallobe epilepsy, some surviving neurons in the boundary

663

664 A. MUNOZ ET AL.

between the subiculum and CA1 lack perisomatic inner-vation, whereas others are hyperinnervated by abnormallydense axons of basket and chandelier cells (Arellano et al.,2004), two key GABA-ergic interneurons controlling theexcitability of pyramidal neurons (Freund and Buzsaki,1996; DeFelipe, 1999). In rats, sustained interictal activ-ity in hippocampal slices downregulates KCC2 mRNAand protein expression in CA1 pyramidal neurons (Riveraet al., 2004); further, KCC2 in the hippocampus is down-regulated after kindling-induced seizures in vivo (Riveraet al., 2002). Amygdala kindling induces selective upreg-ulation of mRNA for NKCC1 contributing to neuronalhyperexcitability (Okabe et al., 2002). Increased expres-sion of NKCC1 precedes hippocampal seizures in ger-bils (Kang et al., 2002). Quantitative RT-PCR analyses ofsurgical specimens taken from the subiculum of patientswith drug-resistant temporal lobe epilepsy reveal upregu-lation of NKCC1 mRNA and down-regulation of KCC2mRNA (Palma et al., 2006). Pharmacological inhibitionof cation-chloride cotransporters (CCCs) by loop diuret-ics blocks epileptiform activity in hippocampal slices dueto NKCC blockade (Schwartzkroin et al., 1998; Hochmanet al., 1999; Hochman and Schwartzkroin, 2000); CCCsinhibitors also have anticonvulsant properties in humansand rodents (Hesdorffer et al., 2001; Fukuda, 2005; Dzhalaet al., 2005).

In the present study, we characterize the expression ofKCC2 and putative NKCC1 proteins in normal neocortexand epileptic temporal lobe of patients with hippocampalsclerosis. The results provide morphological data support-ing the hypothesis that alterations in the relative expressionof KCC2 and NKCC1 in neurons having altered GABA-ergic innervation may contribute to epileptiform activityin the subiculum of patients with hippocampal sclerosis.Some of these results have appeared published in abstractform (Munoz et al., 2004).

METHODS

In the present study, we used adult human brain tissue(n = 14) from two sources: autopsies (kindly suppliedby Dr. R. Alcaraz, Forensic Pathology Service, BasqueInstitute of Legal Medicine, Bilbao, Spain) and postop-erative tissue from patients (Neurosurgery Service, Hos-pital de la Princesa, Madrid, Spain). The autopsy tissuewas obtained at 2–3 h postmortem, from three normalmales who died in traffic accidents (aged 23, 49, and 69years). The human tissue obtained by biopsy through sur-gical intervention was from the temporal neocortex andhippocampal formation of 11 patients (H48, H65, H109,H123, H225, H239, H240, H241, H242, H247, and H248)diagnosed with intractable mesial temporal lobe epilepsy(sex: 3 males, 8 females; mean age and range: 33.8, 21–65years; mean age and range of onset: 12.9, 6–18 years; meanand range of duration: 23.9, 4–50 years). According to the

Helsinki Declaration, the patient’s consent was obtainedin all cases (British Medical Journal, 302: 1194, 1991)and all the protocols were approved by the InstitutionalEthical Committee (Protocols 4/2002 and 14/2002; Hos-pital de la Princesa, Madrid, Spain). Video-EEG recordingwas performed through electrodes located in the scalp andbilaterally in the foramen ovale to locate the epileptic foci.Epileptogenic regions were further identified at the timeof surgery through subdural electrocorticographic (ECoG)recordings with a grid of 4 × 5 electrodes and a strip offour electrodes embedded in Sylastic, with a 1.2 mm in di-ameter and 1-cm center-to-center interelectrode distance(Add-Tech, Medical Instrument Cooperation, Racine, WI,U.S.A.). These electrodes were placed directly over theexposed lateral temporal neocortex or uncus and parahip-pocampal gyrus, respectively. Recordings were performedwith a 32-channel Easy EEG II (Cadwell, Kennewick, WA,U.S.A.) and sampled at 400 Hz with a bandwidth of 1–70 Hz over a minimum period of 20 min. The electrodesthat recorded spikes (<80 ms) or sharp waves (80–200ms) with a mean frequency greater than 1 spike/minuteidentified the spiking areas. Nonspiking areas were de-fined as those in which no spikes, sharp waves, or slowactivity were detected by the electrodes. Photographs ofthe electrode locations were taken before removal of thegrid and the spiking and nonspiking areas were identifiedprior to tissue excision. Tailored temporal lobectomy plusamigdalohippocampectomy were performed under elec-trocorticography guidance in all cases. After surgery, thespiking and nonspiking areas of the lateral neocortex andmesial structures were subjected to standard neuropatho-logical assessment. Hippocampal sclerosis was observedin eight of the 11 patients (H48, H109, H123, H225, H239,H240, H241, and H247). It was characterized by neuronalloss, granule cell dispersion, and mossy fiber prolifera-tion in the dentate gyrus, and by neuronal loss and gliosisin varying degree in the stratum pyramidale of the CAfields (cf., Arellano et al., 2004). In the remaining threepatients (H65, H242, and H248) no pathological findingswere observed in the resected tissue and the hippocam-pal formation exhibited apparently normal cytoarchitec-ture. The lateral neocortex was histologically normal in allcases.

Membrane protein identification and deglycosylationThe normal (nonspiking) areas of the lateral neocor-

tex of patient H225 (female, 49-years old) were used forprotein extraction. In addition, we used neocortical andhippocampal tissue from three adult Wistar rats sacrificedwith an overdose of pentobarbital. Membranes were pre-pared from freshly isolated rat neocortex and hippocam-pus, and from normal human neocortex using differentialcentrifugation. Rat and human tissue were each homog-enized, with the aid of a glass homogenizer, in a buffersolution (1 ml per 3 g of tissue) containing (in mM): 200

Epilepsia, Vol. 48, No. 4, 2007

CATION-CHLORIDE COTRANSPORTERS IN EPILEPSY 665

sucrose, 10 Tris, 10 HEPES, and 1 EDTA (pH 7.2 at 24◦).The homogenate was centrifuged at 5,800 × g for 10 minat 4◦C. The supernatant was centrifuged at 48,000 × g for30 min at 4◦C. The final pellet was resuspended in 0.5%SDS, 100-mM Tris (pH 7.6), 1% mercaptoethanol, 50-mM EDTA with protease inhibitors and stored at −80◦C.Protein concentration was determined with a protein assaykit (Bio-Rad, Hercules, CA, U.S.A.) using bovine serumalbumin as the standard. For deglycoslyation experiments,20 µg of membrane protein were denatured by boiling 5min in a solution containing 0.5% SDS, 100-mM Tris (pH7.6), 1% mercaptoethanol, 50-mM EDTA and proteaseinhibitors. Membranes were incubated overnight at 37◦Cin 100 µl of 0.16% SDS, 0.7% Nonidet P 40, 100-mMTris (pH 7.6), 1% mercaptoethanol, 50-mM EDTA, pro-tease inhibitors, and 1 unit of N-glycosidase F (Roche,Mannheim, Germany). Enzymatic treatment was termi-nated by addition of electrophoresis sample buffer (seebelow). Control samples were processed similarly but in-cubation was carried out in the absence of N-glycosidaseF. Prestained molecular weight markers (New EnglandBioLabs, Beverly, MA, U.S.A.) and membrane proteinsamples were boiled in sample buffer (2% SDS, 13-mMTris (pH 6.8), 10% glycerol, 0.1-M DTT (dithiothre-itol) and 0.002% bromophenol blue] and then separatedby SDS-polyacrylamide Gel Electrophoresis and trans-ferred to polyvinylidene difluoride (PVDF) membranes(Immobilon P, Millipore, Billerica, MA, U.S.A.), using aMini-Protean System (Bio-Rad) in transfer buffer [192-mM glycine, 25-mM Tris (pH 8.3) and 15% methanol].The PVDF membrane was blocked in Tris-buffered saline(TBS)-milk [7% nonfat dry milk and 0.05% Tween-20 inTBS (pH 7.4)] for 1 h and then incubated overnight at4◦C in the same solution with the addition of T4 mousemonoclonal anti-NKCC (dilution 1:200) antibodies or rab-bit anti-KCC2 (dilution 1:200) affinity purified antibod-ies. After three 10-min washes in TBS-Tween the mem-brane was incubated with secondary antibody (horseradishperoxidase-conjugated goat anti-mouse or anti-rabbit IgG,respectively; Jackson ImmunoResearch, West Grove, PA,U.S.A.) for 2 h at 24◦C in TBS-Tween. After three washesin TBS, bound antibody was detected using an enhancedchemiluminescence assay (ECL, Amersham Biosciences,Buckinghamshire, United Kingdom).

ImmunocytochemistrySmall blocks of tissue were analyzed from the rostro-

caudal extent of the hippocampal formation of autopsybrains and from the posterior amygdala, the anterior por-tion of the hippocampus (1–3 cm) and the adjacent cortexfrom biopsies. The tissue was initially fixed by immersionin a cold solution of 4% paraformaldehyde in 0.1-M phos-phate buffer pH 7.4 (PB) for 24–36 h. Serial sections werecut on a vibratome (100-µm thick) from both autopsy and

biopsy material and processed for immunoperoxidase orimmunofluorescent labeling.

Immunoperoxidase experimentsSections from the hippocampal formation were pro-

cessed by batches with standard immunocytochemicaltechniques using the following affinity purified antibod-ies: mouse monoclonal antibody (mAb) T4, for detectionof NKCC (dilution 1:1,000); rabbit polyclonal anti-KCC2(dilution 1:1,000, a gift from Dr. J. Payne), mouse-anti-neuron-specific nuclear protein (NeuN) (dilution 1:2,000,Chemicon. Temecula, CA, U.S.A.) and mouse-anti-PV(dilution 1:4,000) or rabbit anti-PV (dilution 1:4,000)from Swant (Bellinzona, Switzerland). The sections werethen processed using the avidin-biotin method, with theappropriate secondary biotinylated antibodies at a dilutionof 1:200 (Vector Laboratories, Burlingame, CA, U.S.A.),and the Vectastain ABC immunoperoxidase kit (Vector)with DAB (Sigma-Aldrich, St. Louis, MO, U.S.A.) as achromogen. The sections were dehydrated, cleared withxylene, and coverslipped. Adjacent sections stained withthionine were used to reveal the cytoarchitectonic bordersbetween different areas and layers.

Double immunolabelingSections were double-stained for NKCC and KCC2,

NKCC and PV, or KCC2 and PV, using the same pri-mary antibodies, dilutions and incubation times indicatedabove. To this end, the sections were first incubatedin a solution containing the following combinations ofprimary antibodies: mouse anti-NKCC and rabbit anti-KCC2, mouse anti-NKCC and rabbit anti-PV or rabbitanti-KCC2 and mouse anti-PV. After rinsing in PBS thesections were incubated for 2 h at room temperature in asolution containing goat anti-rabbit and horse anti-mouseantibodies coupled to Alexa 594 or Alexa fluor 488 (di-lution 1:1,000; Molecular Probes, Eugene, OR, U.SA.),respectively. Sections were then washed, mounted in 50%glycerol in PBS and examined in a Leica TCS 4D con-focal laser scanning system attached to a Leitz DMRIBmicroscope equipped with an argon/krypton mixed gaslaser with excitation peaks at 488 nm (for Alexa 488)and 568 nm (for Alexa 594). Fluorescence emission wasrecorded through separate channels. Z-optical sectioningwas performed at 1.5–3-µm intervals, and optical stacksof 10–16 images were used for figures. For all immuno-cytochemical procedures, controls consisted of processingsome sections either after replacing the primary antibodywith preimmune goat or horse serum, after omission of thesecondary antibody, or after replacement of the secondaryantibody with an inappropriate secondary antibody (i.e.,an antibody directed to a species different from the onein which the primary antibody was raised). No significantimmunolabeling was detected under these control condi-tions. The degree of NKCC and KCC2 colocalization indouble-labeled neurons was quantified in sections from


666 A. MUNOZ ET AL.

the hippocampal formation (including the subiculum andthe hippocampus proper, see Fig. 2) of eight patients, fiveof which displayed hippocampal sclerosis (H48, H109,H240, H241, and H247). The other three patients (H65,H242, and H248) had normal cytoarchitecture. The per-centage of colocalization was estimated in a total of 102microscopic fields (62,500 µm2 each) from the subicu-lum, the subiculum/CA1 region, CA1 and CA4. To gener-ate figures, light microscopic images were captured witha digital camera (Olympus DP50) attached to an Olym-pus light microscope. In all cases Adobe Photoshop 7.0software (Adobe Systems Inc., San Jose, CA, U.S.A.) wasused to generate figure plates.

Specificity of the NKCC and KCC2 antibodiesThe NKCC antibody was generated against a fusion

protein fragment encompassing the last 310 residues ofthe carboxy-terminus (S760–S1212) of the human colonicNKCC, and recognizes both NKCC1 and NKCC2 iso-forms (Lytle et al., 1995). The hybridoma culture super-natant containing the monoclonal antibodies T4 was ob-tained from the Development Studies Hybridoma Bankmaintained by the University of Iowa (Department ofBiological Sciences, Iowa City, IA, U.S.A.) under con-tract N01-HD-7-3263 from the National Institute of ChildHealth and Human Development (NICHD). The mono-clonal antibody (mAb) was purified by affinity chromatog-raphy as described previously (Alvarez-Leefmans et al.,2001). The antibody selectivity for NKCC detection hasbeen characterized extensively, and has been shown torecognize NKCC proteins in a wide variety of cell types(Lytle et al., 1995; Maglova et al., 1998; McDaniel and Ly-tle, 1999) including rat hippocampal (Marty et al., 2002)and cultured cortical neurons (Sun and Murali, 1999),dorsal root ganglion cells, sensory axons and Schwanncells (Alvarez-Leefmans et al., 2001), astrocytes (Yanet al., 2001), and cultured oligodendrocytes (Wang etal., 2003). The T4 mAb is not NKCC-isoform specific.This mAb recognizes both NKCC1 and NKCC2 and anysplice variants of these cotransporters preserving epitopespresent in the last 310 residues of the carboxy (C) ter-minus. However, NKCC2 transcript and proteins are notpresent in the brain (Gamba et al., 1994; Delpire andMount, 2002). Hence, T4 immunostaining in the brain islikely to reveal mainly, if not exclusively, NKCC1 and itssplice variants with conserved C-terminus (Randall et al.,1997; Plotkin et al., 1997b; Vibat et al., 2001). The rabbitanti-KCC2 polyclonal antibody was generated against apurified fusion protein (B22) containing a 112-amino acidsegment of the carboxyl terminus of the rat KCC2 (932–1043). The immune antiserum was purified by affinitychromatography. This antibody specifically recognizes aband of approximately 140-kDa glycoprotein detectableonly within the central nervous system and in KCC2 trans-fected HEK-293 cells (Williams et al., 1999).

RESULTS

Characterization of NKCC and KCC2 proteinsin human brain

Western blot analysis of membranes isolated from adulthuman and rat brain revealed that NKCC proteins inhuman temporal neocortex, rat neocortex, and rat hip-pocampus were similar. In all three cases, the NKCC an-tibody recognized either a single broad band or a dou-blet of cotransporter immunoreactivity ranging in massfrom approximately 175–165 kDa (Fig. 1). This is con-sistent with the reported molecular mass of the glyco-sylated NKCC1 protein identified in many tissues, in-cluding nonhuman brain cells (Lytle et al., 1995; Plotkinet al., 1997a, 1997b; Haas and Forbush, 2000; Yan etal., 2001; Alvarez-Leefmans et al., 2001). After deg-lycosylation with N-glycosidase F, the molecular massof the putative NKCC1 protein doublet was reduced inboth human and rat brain to approximately 145–135 kDa(Fig. 1). The reduced molecular mass is in close agree-ment with the size of the core polypeptide (Payne et al.,1995). Western blots of human brain membranes fromtemporal neocortex biopsies using anti-KCC2 antibodyshowed a single broad band of protein centered at ap-proximately 140 kDa. A similar band of immunoreac-tivity was seen in membrane proteins isolated from ratneocortex and hippocampus, indicating that KCC2 hasthe same molecular weight (∼140 kDa) in both species(Fig. 1). Deglycosylation experiments demonstrated thatKCC2 in human and rat brain tissue is an N-linkedglycoprotein, given that it migrates to a broad band

FIG. 1. Western Blot analysis of membrane proteins preparedfrom human temporal neocortex, rat neocortex and hippocampus,and HEK-293 cells immunostained with T4 monoclonal antibodyagainst NKCC and with polyclonal KCC2 antibody. Membraneswere incubated with (+) or without (−) N-glycosylase F overnightat 37◦C. Note that both NKCC and KCC2 proteins from humanneocortex migrate on SDS-polyacrylamide gel electrophoresis tosimilar distances than those from rat cortex, indicating that theyhave the same molecular weights. Right panel shows controls inwhich primary antibodies were omitted.



approximately 120-kDa following treatment with N-Glycosidase F. This core approximately 120-kDa KCC2protein is similar in size to that predicted from the cDNA(Payne et al., 1996).

Immunolocalization of NKCC and KCC2 in humansubiculum and hippocampus

The distribution of KCC2 and putative NKCC1 was vi-sualized by immunostaining in human cortical tissue ob-tained from three autopsies and biopsies from 11 epilepticpatients. Of the 11 epileptic patients, eight showed typical

FIG. 2. Human hippocampal formationand subicular regions in a temporal lobeepileptic patient (case H123). Neu-N-immunostained section. The figure il-lustrates the analyzed regions and thenomenclature used in the present study.Low (A) and high (B,E) magnificationphotomicrographs. Arrows in A indicateareas of massive neuronal loss in theCA1 field (sclerotic region). The boxedareas in A are shown at a higher mag-nification in B, C, D, and E, respectively.(B) Border region between CA2 and scle-rotic CA1. (C and D) Transitional areabetween the CA1 and the subiculum, re-ferred to as Subiculum/CA1. (E) Subicu-lum with its characteristic clusters of pyra-midal cells (arrows). Scale bar in E is 665µm for A, and 200 µm for B–E. CA1–CA4,hippocampal fields; DG, dentate gyrus;Sub, subiculum; Pres, presubiculum.

histopathological signs of hippocampal sclerosis, and theother three exhibited normal cytoarchitecture throughoutthe hippocampal formation. The degree of damage in thehippocampus of the patients with hippocampal sclerosisvaried; some regions showed gliosis and a virtual lack ofneurons, but others showed no significant neuronal loss(cf. Arellano et al., 2004). The nomenclature used to referto each region in the present study is shown in Fig. 2. Thenormal looking areas are all referred to as “nonscleroticregions.” Since the transition between the subiculum andCA1 is gradual and diffuse, the transitional region is re-ferred to here as “subiculum/CA1.” The subiculum and


668 A. MUNOZ ET AL.

subiculum/CA1 regions are of particular interest becausethey correspond to areas from which interictal-like activityhas been recorded in temporal lobe slices from epilepticpatients with hippocampal sclerosis (Cohen et al., 2002).The areas showing clear neuronal loss and gliosis in CA1will be referred to as “sclerotic regions.” The transitionalareas between sclerotic and nonsclerotic regions will bereferred to as “border regions.” The latter regions corre-sponded to the limits between CA2 and the sclerotic CA1(Fig. 2B), and the subiculum/CA1 region (Fig. 2C).

NKCC-immunoreactivityThe pattern of NKCC immunostaining revealed by the

T4 mAb in human hippocampal tissue from autopsy and

FIG. 3. (A–F) Colocalization of NKCC and KCC2 in the subiculum and in the hippocampus. The confocal images were obtained fromthe same section and microscopic field illustrating double labeling with NKCC and KCC2 antibodies in the subiculum/CA1 region (A–C)and in the CA4 field (D–F) of the hippocampus. NKCC-immunoreactive neurons are visualized in green (A and D), while red images(B and E) show KCC2-immunoreactive elements. C and F were obtained after combining images A and B, and D and E, respectively.Solid arrows indicate examples of pyramidal neurons that colocalize NKCC and KCC2. Note in A–C that some neurons showing diffusehomogeneous staining throughout the cytoplasm of the soma and proximal dendrites (open arrows; which represent the vast majority oflabeled neurons) or an intense and sometimes irregularly shaped pattern (asterisk) of NKCC immunostaining do not express KCC2. Onthe contrary, some KCC2-immunoreactive neurons lack NKCC immunostaining (open arrows in D–F). (G–I) Parvalbumin-immunoreactivebasket formations innervating NKCC-immunoreactive pyramidal cells in the subiculum/CA1. The confocal images were obtained from thesame section and field taken from the subiculm/CA1 region of patient H109, illustrating the innervation of NKCC-immunoreactive neurons(green) by PV-immunoreactive terminals (red). I was obtained after combining images G and H. The PV-immunoreactive hypertrophicbasket formations innervate the cell bodies of neurons expressing NKCC (arrows in G–I). However, some NKCC-immunoreactive pyramidalneurons (asterisks in G–I) lacked perisomatic innervation by PV-immunoreactive terminals. Images A–C represent stacks of 10 opticalsections obtained at steps of 1.74 µm in the z axis (total: 16 µm). Images D-F were obtained from a single optical section of 1 µm in thez axis. Images G–I represent stacks of 10 optical sections obtained at steps of 1.53 µm in the z axis (total 13.8 µm). Scale bar: 30 µm.

biopsy material was very similar. NKCC immunoreactiv-ity was found in the granule cells of the dentate gyrus andin pyramidal and nonpyramidal neurons of the subiculum,and in the stratum pyramidale of all CA regions, localizedin the neuronal somata and proximal dendritic processes(Figs. 3A, D, H). Some scattered NKCC-immunoreactiveneurons were also found in the strata oriens, radiatum, andlacunosum-moleculare in the hippocampus. The stratumpyramidale showed the highest density of immunoreactivecells throughout different CA fields. In addition, abundantsmall rounded NKCC-immunoreactive somata of putativeglial cells were found in both the white and grey matterof the hippocampus. In patients with hippocampal scle-rosis, the sclerotic regions of the stratum pyramidale of



CA1 were characterized by a significant loss of NKCC-immunoreactive neurons, consistent with the cell loss ob-served in adjacent Nissl-stained or Neu-N-immunostainedserial sections (Fig. 2A). Occasionally some survivingpyramidal and nonpyramidal neurons were found in thesclerotic areas that were NKCC-immunoreactive.

KCC2 immunostainingSections from biopsies probed with the KCC2 anti-

body revealed the presence of numerous, intensely im-munoreactive neurons throughout the subiculum and thestratum pyramidale of the hippocampus proper. How-ever, KCC2 immunostaining was inconsistent in the au-topsy material. Therefore, what follows is a descriptionof the pattern of KCC2 immunoreactivity in the subicu-lum and in the hippocampus proper from epileptic patients(with and without hippocampal sclerosis). Most of KCC2-immunostained neurons were pyramidal cells (Figs. 3B,E), although a few somata of nonpyramidal cells were alsolabeled (open arrow in Fig. 3E). KCC2 immunoreactivitywas distributed in the cell body and in the proximal partof the dendrites. In patients with hippocampal sclerosis,KCC2-immunoreactive neurons were lost in parallel withthe loss of neurons observed in Nissl-stained or Neu-N-immunostained sections of the stratum pyramidale in scle-rotic regions of the CA1. However, some surviving neu-rons in sclerotic areas were also KCC2-immunoreactive.

Colocalization of NKCC and KCC2In the subiculum, subiculum/CA1, CA1, and CA4 of

patients with an undamaged (nonsclerotic) hippocampus,double-labeling experiments with anti-NKCC and anti-KCC2 antibodies revealed a high percentage (>95%)of colocalization of NKCC and KCC2 in neurons (to-tal number of NKCC-positive neurons examined = 251).In patients with hippocampal sclerosis, double-labelingexperiments with anti-NKCC and anti-KCC2 antibodiesrevealed a high percentage of colocalization in nonscle-rotic regions of CA4 (Figs. 3D–F) and subiculum, wheremost neurons (∼93%) coexpressed NKCC and KCC2(total number of NKCC positive neurons examined =224). However, the degree of colocalization of NKCCand KCC2 in neurons located in the subiculum/CA1 andin the sclerotic CA1, was lower (∼80%; total number ofNKCC positive neurons examined = 181) than in nonscle-rotic regions. Therefore, in these latter regions approxi-mately 20% of the NKCC-immunoreactive neurons didnot express KCC2. On the other hand, nearly all (>96%)KCC2-immunoreactive neurons coexpressed NKCC in allanalyzed regions in patients with or without hippocampalsclerosis (Figs. 3A–F).

Parvalbumin immunostainingAntibodies against PV characteristically stained non-

pyramidal cell bodies and dense plexus formed by den-dritic and axonal processes; the latter included the axon

terminals of chandelier and basket cells (i.e., basket for-mations). Consistent with a previous study (Arellanoet al., 2004) that used biopsy material obtained fromepileptic patients with hippocampal sclerosis, we foundalterations in inhibitory circuits that were prominent inthe subiculum/CA1 region and in the sclerotic CA1 region(Figs. 3G–I and 4). These alterations were characterizedby the presence of PV-immunoreactive basket formationsthat were distinctly denser and more complex than thoseseen in normal regions (Figs. 3G and 4A, D, G). Thepresence of these PV-immunoreactive hypertrophic bas-ket formations suggests that some of the neurons in theseregions were hyperinnervated by GABA-ergic interneu-rons. Double-labeling experiments were performed to de-termine whether those neurons hyperinnervated by PV-immunoreactive GABA-ergic terminals expressed NKCCor KCC2. In PV/NKCC double-labeled sections fromthe subiculum/CA1 and the sclerotic CA1 regions, 74%of the PV-immunoreactive hypertrophic basket forma-tions (n = 61) innervated neurons expressing NKCC(Fig. 3I), whereas the remaining 26% innervated non-NKCC-expressing neurons.

As for PV/KCC2 double-labeled sections, 76% of thehypertrophic basket formations analyzed (n = 42) in thesubiculum/CA1 and sclerotic subicular regions innervatedKCC2-immunoreactive neurons (arrows in Figs. 4A–C),whereas the remaining 25% innervated KCC2-negativeneurons (arrows in Figs. 4D–I). Some NKCC and KCC2neurons were virtually devoid of PV-immunoreactive ter-minals (e.g., asterisk in Fig. 3I).

DISCUSSION

The present study demonstrates the expression ofcation-chloride cotransporters (CCCs) KCC2 and putativeNKCC1 in normal neocortex and epileptic temporal lobeof humans with hippocampal sclerosis. Western blot anal-ysis revealed the presence of NKCC and KCC2 proteinsin adult human temporal neocortex in all similar to thosefound in the brain of other vertebrates. Immunolabelingshowed that these CCCs were located in the cell bodiesand proximal dendrites of both pyramidal and nonpyrami-dal cells in the subiculum and in the hippocampus properof patients with temporal lobe epilepsy with or withouthippocampal sclerosis. Furthermore, NKCC is also ex-pressed in glial cells while KCC2 is neuron specific. Inthe normal subiculum, subiculum/CA1, CA1, and CA4regions of epileptic patients more than 95% of the pyra-midal neurons coexpressed NKCC and KCC2. The degreeof NKCC/KCC2 colocalization in the subiculum/CA1 andthe sclerotic CA1 regions of patients with hippocampalsclerosis was lower (∼80%) than in the normal looking re-gions. Therefore, in the subiculum/CA1 region, known toexhibit epileptogenic activity, double immunolabeling of


670 A. MUNOZ ET AL.

FIG. 4. Parvalbumin-immunoreactive bas-ket formations innervating KCC2-immuno-reactive neurons. The confocal images arefrom the same section and field taken fromthe subicum/CA1 regions of patients H48 (A–F) and H109 (G–I), illustrating the innervationpatterns of KCC2-immunoreactive neurons(red) by PV-positive terminals (green). C, F,and I were obtained after combining imagesA and B, D and E, and G and H, respectively.Note that some PV-immunoreactive hyper-trophic basket formations innervate KCC2-positive pyramidal cells (arrows in A–C). InD–I, arrows indicate hypertrophic PV-positivebasket formations innervating the cell body ofneurons devoid of KCC2. In G–I, open arrowpoint to a PV-immunoreactive interneuronthat coexpressed KCC2. Images A–C repre-sent stacks of 12 optical sections obtained atsteps of 3.11 µm in the z axis. Images D–Frepresent stacks of 12 optical sections ob-tained at steps of 2.44 µm in the z axis (total27 µm). Images G–I represent stacks of 16optical sections obtained at steps of 3 µm inthe z axis (total 45 µm). Scale bar: 33 µm forA–C and 23 µm for D–I.

NKCC and KCC2 revealed that approximately 20% of theNKCC-immunoreactive neurons do not express KCC2.

PV immunolabeling confirmed the presence of abnor-mally dense (hypertrophic) basket formations surroundingthe cell bodies and proximal dendrites of pyramidal cellsin the subiculum/CA1 region of patients with hippocampalsclerosis (Arellano et al., 2004).

These PV-immunostained hypertrophic processes areaxon terminals from GABA-ergic interneurons, mainlybasket and chandelier cells. Their presence suggests thatcertain pyramidal neurons in the above areas become hy-perinnervated by GABA-ergic axon terminals from in-terneurons, probably as a consequence of sprouting or re-location of axons from basket cells and other interneuronstargeting pyramidal cells (Arellano et al., 2004). Doubleimmunolabeling for PV and NKCC revealed that most hy-pertrophic basket formations (∼74%) innervated NKCC-immunostained pyramidal cells in the subiculum/CA1 andin the sclerotic CA1 regions. Double immunolabelingfor PV and KCC2 showed that, in the same areas, 76%of the hypertrophic baskets innervated KCC2- immunos-tained neurons, whereas the remaining 24% innervatedcells lacking KCC2.

NKCC and KCC2 proteins of adult humancerebral cortex

Western blot analysis revealed NKCC and KCC2 pro-teins in human temporal neocortex similar in molecular

weight and glycosylation characteristics to those foundin rat neocortex and hippocampus, as shown here and inprevious studies (Plotkin et al., 1997b; Williams et al.,1999; Yan et al., 2001; Marty et al., 2002). In this study,we used only neocortical tissue with no histopathologicalalterations and which showed normal electrical activity ascharacterized by ECoG. Thus, the CCC proteins identifiedby Western blot in this study most likely represent the nor-mal characteristics of expression in the adult human cere-bral neocortex. The NKCC antibody recognized a broadband of cotransporter immunoreactivity at about 170 kDa,consistent with the molecular mass of the glycosylatedprotein (Lytle et al., 1995; Payne et al., 1995; Haas andForbush, 2000). This broad band could be resolved intotwo different components (∼165 and 175 kDa) by reduc-ing film exposure. The NKCC monoclonal antibody usedin the present study (T4) was generated against a fusionprotein fragment encompassing the last 310 residues ofthe C-terminus of the human colonic NKCC (Lytle et al.,1995). The C-terminus displays more than 90% identitybetween NKCC isoforms (Payne et al., 1995), so the T4 an-tibody recognizes both NKCC1 and NKCC2 (Lytle et al.,1995). Since NKCC2 is not expressed in vertebrate brain(Gamba et al., 1994; Ecelbarger et al., 1996; Clayton et al.,1998; Becker et al., 2003; reviewed by Gamba, 2005), weconclude that T4-immunoreactivity in the present materialrepresents NKCC1 and that the doublet may correspondto the full length NKCC1 and a splice variant (see below).



Using a polyclonal antibody directed against a 74-aminoacid peptide located in the C-terminus of NKCC1 thatdoes not recognize NKCC2, a protein doublet band (145–155 KDa) has also been detected in Westerns blots of ratforebrain (Plotkin et al., 1997a, 1997b). Similar NKCC1protein doublets have been observed in Western blots fromtissues other than brain. Moreover, the doublets appear us-ing a variety of anti NKCC antibodies such as T4 (Xu et al.,1994; Liedtke et al., 2001), J3 (Flemmer et al., 2002) andthe polyclonal one directed against a peptide comprising74-amino acid residues of the C-terminus (Kaplan et al.,1996). The most common explanation offered in the litera-ture is that the NKCC1 protein doublets represent differentdegrees of glycosylation of the same protein. Surprisingly,there are no published deglycolyation experiments to testthis commonly held speculation. We found that followingN-deglycosylation the doublet persists although the pro-tein bands decreased in molecular mass. This suggests thatthe doublet in the present study represents two variants ofNKCC1 because NKCC2 transcripts and proteins are notpresent in brain tissue. In mouse and humans there are atleast two alternatively spliced RNA variants of NKCC1.In humans these variants have been named NKCCa andNKCC1b (Vibat et al., 2001). Both splice variants producefunctional NKCC1s. NKCC1b is shorter than NKCC1abecause it lacks 48 bp and is analogous to that describedin mouse, which lacks exon 21 (Randall et al., 1997). Exon21 encodes for a peptide of 16-amino acid residues. Thispeptide would give approximately 2-KDa difference be-tween the two bands. Given the smearing typical of West-ern blots it is difficult to ascertain if the observed doubletsin the present study specifically represent these NKCC1aand b variants. Clearly, this is an important issue that needsto be explored in future work.

We also demonstrate the presence in the human brainof KCC2, the neuronal-specific isoform of the K+-Cl−

cotransporter proteins (Payne et al., 1996). KCC2 isolatedfrom human neocortex is an N-linked glycoprotein with amolecular weight of 140 kDa, similar in size to that fromrat cortex as shown here and in previous reports (Williamset al., 1999). As expected, following treatment with N-glycosidase F the protein band migrates to 125 kDa, thesize of the core protein (Payne et al., 1996).

NKCC and KCC2 are developmentally regulated pro-teins in rodents and humans (Plotkin et al., 1997a; Dzhalaet al., 2005). Neuronal precursors and immature corticalneurons are depolarized by GABA (reviewed by Ben-Ari, 2002). This depolarization produces voltage-sensitiveCa2+ entry that plays a key role in neuronal differenti-ation, growth and maturation (Payne et al., 2003). TheGABA-induced depolarizations ensue because the [Cl−]i

in immature neurons is higher than when in passive equi-librium due to an early expression of NKCC1 (Claytonet al., 1998). As development proceeds, KCC2 expressionincreases and GABA becomes hyperpolarizing, remain-

ing like this in most mature central neurons (Plotkin et al.,1997a; Clayton et al., 1998; Rivera et al., 1999). The fate ofNKCC in mature central neurons is controversial. Someclaim that concomitant with the gradual appearance ofKCC2, NKCC expression decreases reaching either verylow levels (Plotkin et al., 1997a) or total disappearancein adult neurons (Yamada et al., 2004). Others show thatNKCC expression appears early in development, increas-ing gradually until reaching a maximum that is maintainedin adult brain (Clayton et al., 1998; Yan et al., 2001). Ourresults show that NKCC is definitely expressed in adulthuman neocortex.

Implications for Cl− regulation and GABA-ergicfunction in normal and epileptogenic human cortex

We found that most cortical neurons coexpress NKCC1and KCC2 suggesting that the level of intracellular Cl− isdetermined by the functional interaction between thesetwo cotransporters. The presence of a system that ex-trudes Cl− (KCC2) coexisting with another that accumu-lates Cl− (NKCC1) in the same cell suggests that Cl−

is tightly regulated (Gillen and Forbush, 1999). In non-neuronal cells NKCCs and KCCs are reciprocally reg-ulated by negative feedback systems of kinases that arecontrolled by the levels of intracellular Cl− (Russell,2000; Lytle and McManus, 2002; Kahle et al., 2005).In patients with hippocampal sclerosis, 20% of NKCC-expressing pyramidal cells lacked KCC2 in the subiculum/CA1 and sclerotic CA1 regions. The lack of expression ofCl− extrusion via KCC2 along with the concurrent ac-tive transport system that accumulates Cl− in the samecells (NKCC) are likely to result in a higher than pas-sive [Cl−]i which means that the Cl− equilibrium poten-tial (ECl) will be much more positive than Em. This isexpected because unlike in the case of skeletal musclecells, the resting (passive) Cl− permeability of neuronsis relatively low (Alvarez-Leefmans, 1990). GABA sig-naling via A-type receptor channels in these cells willbe depolarizing, due to Cl−-efflux driven by the differ-ence between Em and ECl. This hypothesis is supportedby data from studies in animal models of epilepsy in whichKCC2 mRNA and protein expression is down regulated.Under these conditions the impaired extrusion of Cl− mayleave NKCC1 unrestrained, leading to permanent positiveshifts in ECl underlying and therefore GABAA depolariz-ing responses (Deisz, 2002; Rivera et al., 2002; Khalilovet al., 2003). In fact, it has been shown that a completeor partial disruption of KCC2 gene expression results insevere motor deficits, hippocampal hyperexcitability andfrequent generalized seizures (Hubner et al., 2001; Wooet al., 2002; Rivera et al., 2004). In addition, it has beenshown that Cl− accumulation and GABA-induced depo-larizations are abolished in NKCC1 knockout mice (Sunget al., 2000) and the inhibition of NKCC blocks sponta-neous epileptiform activity (Hochman et al., 1995, 1999;


672 A. MUNOZ ET AL.

Schwartzkroin et al., 1998; Hochman and Schwartzkroin,2000; Fukuda, 2005). Thus, we hypothesize that, in tempo-ral lobe epileptic patients with hippocampal sclerosis, thelack of KCC2 in NKCC1-expressing cells may contributeto the depolarizing responses induced by GABA-ergic sig-naling through GABAA receptors in neurons within thesubiculum and its transitional region with CA1 as re-ported previously (Cohen et al., 2002). These cells dis-charge interictal-like bursts and presumably act as pace-makers in generating interictal synchrony (Cohen et al.,2002, 2003). Remodeling of GABA-ergic circuits in thesubiculum/CA1 and sclerotic CA1 regions in epilepticpatients include the hyperinnervation of some pyrami-dal cells by PV-positive GABA-ergic basket formations(Arellano et al., 2004). We found that around 25% of PV-positive hypertrophic basket formations innervated pyra-midal cells lacking KCC2, whereas most of hyperinner-vated pyramidal cells expressed putative NKCC1. Thus, itis likely that some hypertrophic GABA-ergic basket for-mations innervate pyramidal cells that express NKCC1but lack KCC2. In conclusion, the findings described inthe present study demonstrate: (1) the presence of KCC2and putative NKCC1 proteins in adult human brain inall similar to those found in rat cerebral cortex; (2) thatin patients with hippocampal sclerosis, 20% of NKCC-immunoreactive neurons in the subiculum/CA1 do notexpress KCC2. In the same region, approximately 75%of hypertrophic GABA-ergic terminals innervate NKCC-positive neurons whereas 25% innervate KCC2-negativecells. Altogether these findings represent a morphologicalsubstrate that could explain the anomalous GABA depo-larizing responses found in pyramidal cells by Cohen et al.(2002). These responses participate in the generation ofseizure activity in the human epileptogenic sclerotic hip-pocampus. Consistent with our results and conclusionsare recent observations (Dzhala et al., 2005) showing thatepileptiform activity in perinatal humans and rodents iscorrelated to relatively high expression of NKCC1 withrespect to KCC2.

Acknowledgments: This work was supported by the Min-isterio de Ciencia y Tecnologıa grants BFI 2003-01018 andBFU2006-03855 to A.M. and BFI 2003-02745 and BFU2006-13395 to J. de F., and by the NINDS-NIH grant NS29227to F.J.A-L. Part of this work was carried out while F.J.A.-L.was a faculty member of the Department of Pharmacobiology,Cinvestav-IPN, Mexico, and of the Department of Neurobiology,National Institute of Psychiatry, Mexico. We thank Dr. J. Paynefor providing the KCC2 antisera.

REFERENCES

Alvarez-Leefmans FJ. (1990) In Alvarez-Leefmans FJ, Russell JM (Eds)Chloride channels and carriers in nerve, muscle, and glial cells.Plenum Publishing Corp, New York, pp 109–158.

Alvarez-Leefmans FJ, Leon-Olea M, Mendoza-Sotelo J, Alvarez FJ, An-ton B, Garduno R. (2001) Immunolocalization of the Na(+)-K(+)-2Cl(−) cotransporter in peripheral nervous tissue of vertebrates. Neu-roscience 104:569–582.

Arellano JI, Munoz A, Ballesteros-Yanez I, Sola RG, DeFelipe J. (2004)Histopathology and reorganization of chandelier cells in the humanepileptic sclerotic hippocampus. Brain 127:45–64.

Becker M, Nothwang HG, Friauf E. (2003) Differential expression pat-tern of chloride transporters NCC, NKCC2, KCC1, KCC3, KCC4,and AE3 in the developing rat auditory brainstem. Cell Tissue Re-search 312:155–165.

Ben-Ari Y. (2002) Excitatory actions of GABA during development: thenature of the nurture. Nature Reviews Neuroscience 9:728–739.

Clayton GH, Owens GC, Wolff JS, Smith RL. (1998) Ontogeny of cation-Cl− cotransporter expression in rat neocortex. Brain Research De-velopment Brain Research 109:281–292.

Cohen I, Navarro V, Clemenceau S, Baulac M, Miles R. (2002) On theorigin of interictal activity in human temporal lobe epilepsy in vitro.Science 298:1418–1421.

Cohen I, Navarro V, Le Duigou C, Miles R. (2003) Mesial temporallobe epilepsy: a pathological replay of developmental mechanisms?Biologie Cellulaire 95:329–333.

DeFelipe J. (1999) Chandelier cells and epilepsy. Brain 122:1807–1822.Deisz RA. (2002) Cellular mechanisms of pharmacoresistance in slices

from epilepsy surgery. Novartis Foundation Symposium 243:186–199.

Delpire E, Mount DB. (2002) Human and murine phenotypes associ-ated with defects in cation-chloride cotransport. Annual Review ofPhysiology 64:803–843.

Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, BenkeTA, Delpire E, Jensen FE, Staley KJ. (2005) NKCC1 transporter fa-cilitates seizures in the developing brain. Nature Medicine 11:1205–1213.

Ecelbarger CA, Terris J, Hoyer JR, Nielsen S, Wade JB, KnepperMA. (1996) Localization and regulation of the rat renal Na(+)-K(+)-2 Cl− cotransporter, BSC-1. American Journal of Physiology271:F619–F628.

Flemmer AW, Gimenez I, Dowd BF, Darman RB, Forbush B. (2002)Activation of the Na-K-Cl cotransporter NKCC1 detected witha phospho-specific antibody. Journal of Biological Chemistry277:37551–37558.

Freund TF, Buzsaki G. (1996) Interneurons of the hippocampus. Hip-pocampus 6:347–470.

Fukuda A. (2005) Diuretic soothes seizures in newborns. NatureMedicine 11:1153–1154.

Gamba G. (2005) Molecular physiology and pathophysiology of elec-troneutral cation-chloride cotransporters. Physiological Reviews85:423–493.

Gamba G, Miyanoshita A, Lombardi M. (1994) Molecular cloning,primary structure, and characterization of two members of themammalian electroneutral sodium-(potassium)-chloride cotrans-porter family expressed in kidney. Journal of Biological Chemistry269:17713–17722.

Gillen CM, Forbush BI II. (1999) Functional interaction of the K-Clcotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293cells. American Journal of Physiology 276:C328–C336.

Haas M, Forbush BI II. (2000) The Na-K-Cl cotransporter of secretoryepithelia. Annual Review of Physiology 62:515–534.

Hesdorffer DC, Stables JP, Hauser WA, Annegers JF, Cascino G. (2001)Are certain diuretics also anticonvulsants? Annals of Neurology50:458–462.

Hochman DW, Baraban SC, Owens JW, Schwartzkroin PA. (1995) Dis-sociation of synchronization and excitability in furosemide blockadeof epileptiform activity. Science 270:99–102.

Hochman DW, D’Ambrosio R, Janigro D, Schwartzkroin PA. (1999)Extracellular chloride and the maintenance of spontaneous epilepti-form activity in rat hippocampal slices. Journal of Neurophysiology81:49–59.

Hochman DW, Schwartzkroin PA. (2000) Chloride-cotransport blockadedesynchronizes neuronal discharge in the “epileptic” hippocampalslice. Journal of Neurophysiology 83:406–417.

Honavar M, Meldrum BS. (1997) Epilepsy. In Graham DI, Lantos PL,(Eds). Greenfield’s neuropathology. Arnold, London, pp. 931–971.

Hubner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K,Jentsch TJ. (2001) Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron 30:515–524.



Khalilov I, Holmes GL, Ben Ari Y. (2003) In vitro formation of a sec-ondary epileptogenic mirror focus by interhippocampal propagationof seizures. Nature Neuroscience 6:1079–1085.

Kahle KT, Rinehart J, De los Heros P, Louvi A, Meade P, Vazquez N,Hebert SC, Gamba G, Gimenez I, Lifton RP. (2005) WNK3 modu-lates transport of Cl− in and out of cells: implications for control ofcell volume and neuronal excitability. Proceedings of the NationalAcademy of Sciences of the United States of America 102:16783–16788.

Kang TC, An SJ, Park SK, Hwang IK, Yoon DK, Shin HS, Won MH.(2002) Changes in Na(+)-K(+)-Cl(−) cotransporter immunoreac-tivity in the gerbil hippocampus following transient ischemia. Neu-roscience Research 44:249–254.

Kaplan MR, Plotkin MD, Brown D, Hebert SC, Delpire E. (1996) Expres-sion of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminalinner medullary collecting duct, the glomerular and extraglomerularmesangium, and the glomerular afferent arteriole. Journal of ClinicalInvestigation 98:723–730.

Liedtke CM, Cody D, Cole TS. (2001) Differential regulation of Cl−transport proteins by PKC in Calu-3 cells. American Journal of Phys-iology. Lung Cellular and Molecular Physiology 280:L739–L747.

Lytle C, McManus T. (2002) Coordinate modulation of Na-K-2Cl co-transport and K-Cl cotransport by cell volume and chloride. Ameri-can Journal of Physiology. Cell Physiology 283:C1422–C1431.

Lytle C, Xu JC, Biemesderfer D, Forbush BI II. (1995) Distribution anddiversity of Na-K-Cl cotransport proteins: a study with monoclonalantibodies. American Journal of Physiology 269:C1496–C1505.

Maglova LM, Crowe WE, Smith PR, Altamirano AA, Russell JM.(1998) Na+-K+- Cl−cotransport in human fibroblasts is inhib-ited by cytomegalovirus infection. American Journal of Physiology275:C1330–C1341.

Marty S, Wehrle R, Alvarez-Leefmans FJ, Gasnier B, Sotelo C. (2002)Postnatal maturation of Na+, K+, 2 Cl− cotransporter expression andinhibitory synaptogenesis in the rat hippocampus: an immunocyto-chemical analysis. European Journal of Neuroscience 15:233–245.

McDaniel N, Lytle C. (1999) Parietal cells express high levels of Na-K-2Cl cotransporter on migrating into the gastric gland neck. AmericanJournal of Physiology 276:G1273–G1278.

Munoz A, Mendez P, Alvarez-Leefmans FJ, DeFelipe J. (2004) Alteredexpression of NKCC and KCC2 transporters and GABAergic inner-vation in human epileptic hippocampus. 34th Annual Meeting of theSociety for Neuroscience. Abstract 567.6 Epilepsy, Basic Mecha-nisms V San Diego, California.

Okabe A, Ohno K, Toyoda H, Yokokura M, Sato K, Fukuda A. (2002)Amygdala kindling induces upregulation of mRNA for NKCC1, aNa(+), K(+)-2Cl(−) cotransporter, in the rat piriform cortex. Neu-roscience Research 44:225–229.

Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, RagozzinoD, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F. (2006)Anomalous levels of Cl− transporters in the hippocampal subiculumfrom temporal lobe epilepsy patients make GABA excitatory. Pro-ceedings of the National Academy of Sciences of the United Statesof America 103:8465–8468.

Payne JA, Rivera C, Voipio J, Kaila K. (2003) Cation-chloride co-transporters in neuronal communication, development and trauma.Trends in Neuroscience 26:199–206.

Payne JA, Stevenson TJ, Donaldson LF. (1996) Molecular characteriza-tion of a putative K-Cl cotransporter in rat brain. A neuronal-specificisoform. Journal of Biological Chemistry 271:16245–16252.

Payne JA, Xu JC, Haas M, Lytle CY, Ward D, Forbush BI II. (1995) Pri-mary structure, functional expression, and chromosomal localizationof the bumetanide-sensitive Na-K-Cl cotransporter in human colon.Journal of Biological Chemistry 270:17977–17985.

Plotkin MD, Snyder EY, Hebert SC, Delpire E. (1997a) Expression ofthe Na-K-2Cl cotransporter is developmentally regulated in postnatalrat brains: a possible mechanism underlying GABA’s excitatory rolein immature brain. Journal of Neurobiology 33:781–795.

Plotkin MD, Kaplan MR, Peterson LN, Gullans SR, Hebert SC, DelpireE. (1997b) Expression of the Na+-K+-2Cl− cotransporter BSC2in the nervous system. American Journal of Physiology 272:C173–C183.

Randall J, Thorne T, Delpire E. (1997) Partial cloning and characteri-zation of Slc12a2: the gene encoding the secretory Na+-K+-2Cl−cotransporter. American Journal of Physiology 273:C1267–C1277.

Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, NanobashviliA, Kokaia Z, Airaksinen MS, Voipio J, Kaila K, Saarma M. (2002)BDNF-induced TrkB activation downregulates the K+-Cl− cotrans-porter KCC2 and impairs neuronal Cl− extrusion. Journal of CellBiology 159:747–752.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K,Pirvola U, Saarma M, Kaila K. (1999) The K+/Cl− co-transporterKCC2 renders GABA hyperpolarizing during neuronal maturation.Nature 397:251–255.

Rivera C, Voipio J, Thomas-Crusells J, Li H, Emri Z, Sipila S, Payne JA,Minichiello L, Saarma M, Kaila K. (2004) Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporterKCC2. Journal of Neuroscience 24:4683–4691.

Russell JM. (2000) Sodium-potassium-chloride cotransport. Physiolog-ical Reviews 80:211–276.

Schwartzkroin PA, Baraban SC, Hochman DW. (1998) Osmolarity, ionicflux, and changes in brain excitability. Epilepsy Research 32:275–285.

Sun D, Murali SG. (1999) Na+-K+-2Cl− cotransporter in immaturecortical neurons: A role in intracellular Cl− regulation. Journal ofNeurophysiology 81:1939–1948.

Sung KW, Kirvy M, McDonald MP, Lovinger DM, Delpire E. (2000)Abnormal GABAA receptor-mediated currents in dorsal root gan-glion neurons isolated from Na-K-2Cl cootransporter null-mice.Journal of Neuroscience 20:7531–7538

Wang H, Yan Y, Kintner DB, Lytle C, Sun D. (2003) GABA-mediatedtrophic effect on oligodendrocytes requires Na-K-2Cl cotransportactivity. Journal of Neurophysiology 90:1257–1265.

Williams JR, Sharp JW, Kumari VG, Wilson M, Payne JA. (1999)The neuron-specific K-Cl cotransporter, KCC2. Antibody develop-ment and initial characterization of the protein. Journal of BiologicalChemistry 274:12656–12664.

Woo NS, Lu J, England R, McClellan R, Dufour S, Mount DB, DeutchAY, Lovinger DM, Delpire E. (2002) Hyperexcitability and epilepsyassociated with disruption of the mouse neuronal-specific K-Cl co-transporter gene. Hippocampus 12:258–268.

Vibat CR, Holland MJ, Kang JJ, Putney LK, O’Donnell ME. (2001)Quantitation of Na+-K+-2Cl− cotransport splice variants in humantissues using kinetic polymerase chain reaction. Analytical Biochem-istry 298:218–230.

Xu JC, Lytle C, Zhu TT, Payne JA, Benz E Jr, Forbush B 3rd. (1994)Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. Proceedings of the NationalAcademy of Sciences of the United States of America 91:2201–2205.

Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A. (2004)Cl− uptake promoting depolarizing GABA actions in immature ratneocortical neurones is mediated by NKCC1. Journal of Physiology557:829–841.

Yan Y, Dempsey RJ, Sun D. (2001) Expression of Na(+)-K(+)-Cl(−)cotransporter in rat brain during development and its localization inmature astrocytes. Brain Research 911:43–55.


Cation-Chloride Cotransporters and GABA-ergic Innervation in the Human Epileptic Hippocampus

Documents

Transcript of Cation-Chloride Cotransporters and GABA-ergic Innervation in the Human Epileptic Hippocampus