•  Epilepsy;
  • Subiculum;
  • Hippocampal sclerosis;
  • Inhibition;
  • NKCC1;
  • KCC2


  1. Top of page
  2. Abstract
  6. Acknowledgments

Summary:  Intracellular chloride concentration, [Cl]i, determines the polarity of GABAA-induced neuronal Cl currents. In neurons, [Cl]i is set by the activity of Na+, K+, 2Cl cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl in the cell, and Cl extruding K+, Cl cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex.

NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.

Hippocampal sclerosis, a common feature of patients with temporal lobe epilepsy, is characterized by cell loss and gliosis in various regions of the hippocampal formation, notably CA1, leaving the subiculum mostly intact (Honavar and Meldrum, 1997). Electrophysiological recordings from human temporal lobe slices from epileptic patients with hippocampal sclerosis revealed spontaneous synchronous interictal-like discharges, similar to those recorded in intracranial electroencephalograms from the same patients (Cohen et al., 2002). The interictal-like discharges were initiated in the subiculum and in its transitional area with CA1. The subicular circuit displaying these interictal-like discharges includes interneurons and a subgroup of pyramidal cells that are depolarized by GABA released from interneurons. This depolarizing GABA-ergic signaling is likely to be involved in the generation of these spontaneous interictal discharges in the temporal lobe of epileptic patients with hippocampal sclerosis (Cohen et al., 2002; Deisz, 2002). The direction and magnitude of GABA-induced Cl currents through GABAA receptors is determined by the intracellular [Cl] resulting mostly from the functional expression of the Na+-K+-2Cl cotransporters (NKCCs) that accumulate Cl inside the cells, and the Cl extruding K+-Cl cotransporters (KCCs) (Alvarez-Leefmans, 1990; Alvarez-Leefmans et al., 2001; Delpire and Mount, 2002; Gamba, 2005). Alterations in the balance of NKCCs and KCCs activity may determine the switch from a hyperpolarizing to a depolarizing effect of GABA, thereby contributing to epileptogenesis in human hippocampal formation (Cohen et al., 2003; Fukuda, 2005; Dzhala et al., 2005).

Various lines of evidence correlate epileptogenesis with altered functional expression of NKCC and KCC transporters. Accordingly, deletion of KCC2 gene expression in mice causes hyperexcitability in the hippocampus, generalized seizures, and death shortly after birth (Woo et al., 2002). These changes are accompanied by loss of neurons positive for parvalbumin (PV), which are presumed to be inhibitory GABA-ergic interneurons. In human temporal lobe epilepsy, some surviving neurons in the boundary between the subiculum and CA1 lack perisomatic innervation, whereas others are hyperinnervated by abnormally dense axons of basket and chandelier cells (Arellano et al., 2004), two key GABA-ergic interneurons controlling the excitability of pyramidal neurons (Freund and Buzsaki, 1996; DeFelipe, 1999). In rats, sustained interictal activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons (Rivera et al., 2004); further, KCC2 in the hippocampus is downregulated after kindling-induced seizures in vivo (Rivera et al., 2002). Amygdala kindling induces selective upregulation of mRNA for NKCC1 contributing to neuronal hyperexcitability (Okabe et al., 2002). Increased expression of NKCC1 precedes hippocampal seizures in gerbils (Kang et al., 2002). Quantitative RT-PCR analyses of surgical specimens taken from the subiculum of patients with drug-resistant temporal lobe epilepsy reveal upregulation of NKCC1 mRNA and down-regulation of KCC2 mRNA (Palma et al., 2006). Pharmacological inhibition of cation-chloride cotransporters (CCCs) by loop diuretics blocks epileptiform activity in hippocampal slices due to NKCC blockade (Schwartzkroin et al., 1998; Hochman et al., 1999; Hochman and Schwartzkroin, 2000); CCCs inhibitors also have anticonvulsant properties in humans and rodents (Hesdorffer et al., 2001; Fukuda, 2005; Dzhala et al., 2005).

In the present study, we characterize the expression of KCC2 and putative NKCC1 proteins in normal neocortex and epileptic temporal lobe of patients with hippocampal sclerosis. The results provide morphological data supporting the hypothesis that alterations in the relative expression of KCC2 and NKCC1 in neurons having altered GABA-ergic innervation may contribute to epileptiform activity in the subiculum of patients with hippocampal sclerosis. Some of these results have appeared published in abstract form (Muñoz et al., 2004).


  1. Top of page
  2. Abstract
  6. Acknowledgments

In the present study, we used adult human brain tissue (n = 14) from two sources: autopsies (kindly supplied by Dr. R. Alcaraz, Forensic Pathology Service, Basque Institute of Legal Medicine, Bilbao, Spain) and postoperative tissue from patients (Neurosurgery Service, Hospital de la Princesa, Madrid, Spain). The autopsy tissue was obtained at 2–3 h postmortem, from three normal males who died in traffic accidents (aged 23, 49, and 69 years). The human tissue obtained by biopsy through surgical intervention was from the temporal neocortex and hippocampal 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, 21–65 years; mean age and range of onset: 12.9, 6–18 years; mean and range of duration: 23.9, 4–50 years). According to the Helsinki Declaration, the patient's consent was obtained in all cases (British Medical Journal, 302: 1194, 1991) and all the protocols were approved by the Institutional Ethical Committee (Protocols 4/2002 and 14/2002; Hospital de la Princesa, Madrid, Spain). Video-EEG recording was performed through electrodes located in the scalp and bilaterally in the foramen ovale to locate the epileptic foci. Epileptogenic regions were further identified at the time of surgery through subdural electrocorticographic (ECoG) recordings with a grid of 4 × 5 electrodes and a strip of four electrodes embedded in Sylastic, with a 1.2 mm in diameter and 1-cm center-to-center interelectrode distance (Add-Tech, Medical Instrument Cooperation, Racine, WI, U.S.A.). These electrodes were placed directly over the exposed lateral temporal neocortex or uncus and parahippocampal gyrus, respectively. Recordings were performed with 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 electrodes that recorded spikes (<80 ms) or sharp waves (80–200 ms) with a mean frequency greater than 1 spike/minute identified the spiking areas. Nonspiking areas were defined as those in which no spikes, sharp waves, or slow activity were detected by the electrodes. Photographs of the electrode locations were taken before removal of the grid and the spiking and nonspiking areas were identified prior to tissue excision. Tailored temporal lobectomy plus amigdalohippocampectomy were performed under electrocorticography guidance in all cases. After surgery, the spiking and nonspiking areas of the lateral neocortex and mesial structures were subjected to standard neuropathological assessment. Hippocampal sclerosis was observed in eight of the 11 patients (H48, H109, H123, H225, H239, H240, H241, and H247). It was characterized by neuronal loss, granule cell dispersion, and mossy fiber proliferation in the dentate gyrus, and by neuronal loss and gliosis in varying degree in the stratum pyramidale of the CA fields (cf., Arellano et al., 2004). In the remaining three patients (H65, H242, and H248) no pathological findings were observed in the resected tissue and the hippocampal formation exhibited apparently normal cytoarchitecture. The lateral neocortex was histologically normal in all cases.

Membrane protein identification and deglycosylation

The normal (nonspiking) areas of the lateral neocortex of patient H225 (female, 49-years old) were used for protein extraction. In addition, we used neocortical and hippocampal tissue from three adult Wistar rats sacrificed with an overdose of pentobarbital. Membranes were prepared from freshly isolated rat neocortex and hippocampus, and from normal human neocortex using differential centrifugation. Rat and human tissue were each homogenized, with the aid of a glass homogenizer, in a buffer solution (1 ml per 3 g of tissue) containing (in mM): 200 sucrose, 10 Tris, 10 HEPES, and 1 EDTA (pH 7.2 at 24°). The homogenate was centrifuged at 5,800 ×g for 10 min at 4°C. The supernatant was centrifuged at 48,000 ×g for 30 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 assay kit (Bio-Rad, Hercules, CA, U.S.A.) using bovine serum albumin as the standard. For deglycoslyation experiments, 20 μg of membrane protein were denatured by boiling 5 min in a solution containing 0.5% SDS, 100-mM Tris (pH 7.6), 1% mercaptoethanol, 50-mM EDTA and protease inhibitors. Membranes were incubated overnight at 37°C in 100 μl of 0.16% SDS, 0.7% Nonidet P 40, 100-mM Tris (pH 7.6), 1% mercaptoethanol, 50-mM EDTA, protease inhibitors, and 1 unit of N-glycosidase F (Roche, Mannheim, Germany). Enzymatic treatment was terminated by addition of electrophoresis sample buffer (see below). Control samples were processed similarly but incubation was carried out in the absence of N-glycosidase F. Prestained molecular weight markers (New England BioLabs, Beverly, MA, U.S.A.) and membrane protein samples were boiled in sample buffer (2% SDS, 13-mM Tris (pH 6.8), 10% glycerol, 0.1-M DTT (dithiothreitol) and 0.002% bromophenol blue] and then separated by SDS-polyacrylamide Gel Electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore, Billerica, MA, U.S.A.), using a Mini-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 in TBS (pH 7.4)] for 1 h and then incubated overnight at 4°C in the same solution with the addition of T4 mouse monoclonal anti-NKCC (dilution 1:200) antibodies or rabbit anti-KCC2 (dilution 1:200) affinity purified antibodies. After three 10-min washes in TBS-Tween the membrane was incubated with secondary antibody (horseradish peroxidase-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 washes in TBS, bound antibody was detected using an enhanced chemiluminescence assay (ECL, Amersham Biosciences, Buckinghamshire, United Kingdom).


Small blocks of tissue were analyzed from the rostrocaudal extent of the hippocampal formation of autopsy brains and from the posterior amygdala, the anterior portion of the hippocampus (1–3 cm) and the adjacent cortex from biopsies. The tissue was initially fixed by immersion in a cold solution of 4% paraformaldehyde in 0.1-M phosphate buffer pH 7.4 (PB) for 24–36 h. Serial sections were cut on a vibratome (100-μm thick) from both autopsy and biopsy material and processed for immunoperoxidase or immunofluorescent labeling.

Immunoperoxidase experiments

Sections from the hippocampal formation were processed by batches with standard immunocytochemical techniques using the following affinity purified antibodies: mouse monoclonal antibody (mAb) T4, for detection of 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 were then processed using the avidin-biotin method, with the appropriate secondary biotinylated antibodies at a dilution of 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 a chromogen. The sections were dehydrated, cleared with xylene, and coverslipped. Adjacent sections stained with thionine were used to reveal the cytoarchitectonic borders between different areas and layers.

Double immunolabeling

Sections were double-stained for NKCC and KCC2, NKCC and PV, or KCC2 and PV, using the same primary antibodies, dilutions and incubation times indicated above. To this end, the sections were first incubated in a solution containing the following combinations of primary antibodies: mouse anti-NKCC and rabbit anti-KCC2, mouse anti-NKCC and rabbit anti-PV or rabbit anti-KCC2 and mouse anti-PV. After rinsing in PBS the sections were incubated for 2 h at room temperature in a solution containing goat anti-rabbit and horse anti-mouse antibodies coupled to Alexa 594 or Alexa fluor 488 (dilution 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 confocal laser scanning system attached to a Leitz DMRIB microscope equipped with an argon/krypton mixed gas laser with excitation peaks at 488 nm (for Alexa 488) and 568 nm (for Alexa 594). Fluorescence emission was recorded through separate channels. Z-optical sectioning was performed at 1.5–3-μm intervals, and optical stacks of 10–16 images were used for figures. For all immunocytochemical procedures, controls consisted of processing some sections either after replacing the primary antibody with preimmune goat or horse serum, after omission of the secondary antibody, or after replacement of the secondary antibody with an inappropriate secondary antibody (i.e., an antibody directed to a species different from the one in which the primary antibody was raised). No significant immunolabeling was detected under these control conditions. The degree of NKCC and KCC2 colocalization in double-labeled neurons was quantified in sections from the hippocampal formation (including the subiculum and the hippocampus proper, see Fig. 2) of eight patients, five of which displayed hippocampal sclerosis (H48, H109, H240, H241, and H247). The other three patients (H65, H242, and H248) had normal cytoarchitecture. The percentage of colocalization was estimated in a total of 102 microscopic fields (62,500 μm2 each) from the subiculum, the subiculum/CA1 region, CA1 and CA4. To generate figures, light microscopic images were captured with a digital camera (Olympus DP50) attached to an Olympus light microscope. In all cases Adobe Photoshop 7.0 software (Adobe Systems Inc., San Jose, CA, U.S.A.) was used to generate figure plates.


Figure 2. Human hippocampal formation and subicular regions in a temporal lobe epileptic patient (case H123). Neu-N-immunostained section. The figure illustrates the analyzed regions and the nomenclature used in the present study. Low (A) and high (B,E) magnification photomicrographs. Arrows in A indicate areas of massive neuronal loss in the CA1 field (sclerotic region). The boxed areas in A are shown at a higher magnification in B, C, D, and E, respectively. (B) Border region between CA2 and sclerotic CA1. (C and D) Transitional area between the CA1 and the subiculum, referred to as Subiculum/CA1. (E) Subiculum with its characteristic clusters of pyramidal cells (arrows). Scale bar in E is 665 μm for A, and 200 μm for B–E. CA1–CA4, hippocampal fields; DG, dentate gyrus; Sub, subiculum; Pres, presubiculum.

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Specificity of the NKCC and KCC2 antibodies

The NKCC antibody was generated against a fusion protein fragment encompassing the last 310 residues of the carboxy-terminus (S760–S1212) of the human colonic NKCC, and recognizes both NKCC1 and NKCC2 isoforms (Lytle et al., 1995). The hybridoma culture supernatant containing the monoclonal antibodies T4 was obtained from the Development Studies Hybridoma Bank maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA, U.S.A.) under contract N01-HD-7-3263 from the National Institute of Child Health and Human Development (NICHD). The monoclonal antibody (mAb) was purified by affinity chromatography as described previously (Alvarez-Leefmans et al., 2001). The antibody selectivity for NKCC detection has been characterized extensively, and has been shown to recognize NKCC proteins in a wide variety of cell types (Lytle et al., 1995; Maglova et al., 1998; McDaniel and Lytle, 1999) including rat hippocampal (Marty et al., 2002) and cultured cortical neurons (Sun and Murali, 1999), dorsal root ganglion cells, sensory axons and Schwann cells (Alvarez-Leefmans et al., 2001), astrocytes (Yan et al., 2001), and cultured oligodendrocytes (Wang et al., 2003). The T4 mAb is not NKCC-isoform specific. This mAb recognizes both NKCC1 and NKCC2 and any splice variants of these cotransporters preserving epitopes present in the last 310 residues of the carboxy (C) terminus. However, NKCC2 transcript and proteins are not present in the brain (Gamba et al., 1994; Delpire and Mount, 2002). Hence, T4 immunostaining in the brain is likely to reveal mainly, if not exclusively, NKCC1 and its splice variants with conserved C-terminus (Randall et al., 1997; Plotkin et al., 1997b; Vibat et al., 2001). The rabbit anti-KCC2 polyclonal antibody was generated against a purified fusion protein (B22) containing a 112-amino acid segment of the carboxyl terminus of the rat KCC2 (932–1043). The immune antiserum was purified by affinity chromatography. This antibody specifically recognizes a band of approximately 140-kDa glycoprotein detectable only within the central nervous system and in KCC2 transfected HEK-293 cells (Williams et al., 1999).


  1. Top of page
  2. Abstract
  6. Acknowledgments

Characterization of NKCC and KCC2 proteins in human brain

Western blot analysis of membranes isolated from adult human and rat brain revealed that NKCC proteins in human temporal neocortex, rat neocortex, and rat hippocampus were similar. In all three cases, the NKCC antibody recognized either a single broad band or a doublet of cotransporter immunoreactivity ranging in mass from approximately 175–165 kDa (Fig. 1). This is consistent with the reported molecular mass of the glycosylated NKCC1 protein identified in many tissues, including nonhuman brain cells (Lytle et al., 1995; Plotkin et al., 1997a, 1997b; Haas and Forbush, 2000; Yan et al., 2001; Alvarez-Leefmans et al., 2001). After deglycosylation with N-glycosidase F, the molecular mass of the putative NKCC1 protein doublet was reduced in both human and rat brain to approximately 145–135 kDa (Fig. 1). The reduced molecular mass is in close agreement with the size of the core polypeptide (Payne et al., 1995). Western blots of human brain membranes from temporal neocortex biopsies using anti-KCC2 antibody showed a single broad band of protein centered at approximately 140 kDa. A similar band of immunoreactivity was seen in membrane proteins isolated from rat neocortex and hippocampus, indicating that KCC2 has the same molecular weight (∼140 kDa) in both species (Fig. 1). Deglycosylation experiments demonstrated that KCC2 in human and rat brain tissue is an N-linked glycoprotein, given that it migrates to a broad band approximately 120-kDa following treatment with N-Glycosidase F. This core approximately 120-kDa KCC2 protein is similar in size to that predicted from the cDNA (Payne et al., 1996).


Figure 1. Western Blot analysis of membrane proteins prepared from human temporal neocortex, rat neocortex and hippocampus, and HEK-293 cells immunostained with T4 monoclonal antibody against NKCC and with polyclonal KCC2 antibody. Membranes were incubated with (+) or without (−) N-glycosylase F overnight at 37°C. Note that both NKCC and KCC2 proteins from human neocortex migrate on SDS-polyacrylamide gel electrophoresis to similar distances than those from rat cortex, indicating that they have the same molecular weights. Right panel shows controls in which primary antibodies were omitted.

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Immunolocalization of NKCC and KCC2 in human subiculum and hippocampus

The distribution of KCC2 and putative NKCC1 was visualized by immunostaining in human cortical tissue obtained from three autopsies and biopsies from 11 epileptic patients. Of the 11 epileptic patients, eight showed typical histopathological signs of hippocampal sclerosis, and the other three exhibited normal cytoarchitecture throughout the hippocampal formation. The degree of damage in the hippocampus of the patients with hippocampal sclerosis varied; some regions showed gliosis and a virtual lack of neurons, but others showed no significant neuronal loss (cf. Arellano et al., 2004). The nomenclature used to refer to each region in the present study is shown in Fig. 2. The normal looking areas are all referred to as “nonsclerotic regions.” Since the transition between the subiculum and CA1 is gradual and diffuse, the transitional region is referred to here as “subiculum/CA1.” The subiculum and subiculum/CA1 regions are of particular interest because they correspond to areas from which interictal-like activity has been recorded in temporal lobe slices from epileptic patients with hippocampal sclerosis (Cohen et al., 2002). The areas showing clear neuronal loss and gliosis in CA1 will be referred to as “sclerotic regions.” The transitional areas between sclerotic and nonsclerotic regions will be referred to as “border regions.” The latter regions corresponded to the limits between CA2 and the sclerotic CA1 (Fig. 2B), and the subiculum/CA1 region (Fig. 2C).


The pattern of NKCC immunostaining revealed by the T4 mAb in human hippocampal tissue from autopsy and biopsy material was very similar. NKCC immunoreactivity was found in the granule cells of the dentate gyrus and in pyramidal and nonpyramidal neurons of the subiculum, and in the stratum pyramidale of all CA regions, localized in the neuronal somata and proximal dendritic processes (Figs. 3A, D, H). Some scattered NKCC-immunoreactive neurons were also found in the strata oriens, radiatum, and lacunosum-moleculare in the hippocampus. The stratum pyramidale showed the highest density of immunoreactive cells throughout different CA fields. In addition, abundant small rounded NKCC-immunoreactive somata of putative glial cells were found in both the white and grey matter of the hippocampus. In patients with hippocampal sclerosis, the sclerotic regions of the stratum pyramidale of CA1 were characterized by a significant loss of NKCC-immunoreactive neurons, consistent with the cell loss observed in adjacent Nissl-stained or Neu-N-immunostained serial sections (Fig. 2A). Occasionally some surviving pyramidal and nonpyramidal neurons were found in the sclerotic areas that were NKCC-immunoreactive.


Figure 3. (A–F) Colocalization of NKCC and KCC2 in the subiculum and in the hippocampus. The confocal images were obtained from the 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 diffuse homogeneous staining throughout the cytoplasm of the soma and proximal dendrites (open arrows; which represent the vast majority of labeled neurons) or an intense and sometimes irregularly shaped pattern (asterisk) of NKCC immunostaining do not express KCC2. On the contrary, some KCC2-immunoreactive neurons lack NKCC immunostaining (open arrows in D–F). (G–I) Parvalbumin-immunoreactive basket formations innervating NKCC-immunoreactive pyramidal cells in the subiculum/CA1. The confocal images were obtained from the same 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 hypertrophic basket formations innervate the cell bodies of neurons expressing NKCC (arrows in G–I). However, some NKCC-immunoreactive pyramidal neurons (asterisks in G–I) lacked perisomatic innervation by PV-immunoreactive terminals. Images A–C represent stacks of 10 optical sections 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 the z 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.

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KCC2 immunostaining

Sections from biopsies probed with the KCC2 antibody revealed the presence of numerous, intensely immunoreactive neurons throughout the subiculum and the stratum pyramidale of the hippocampus proper. However, KCC2 immunostaining was inconsistent in the autopsy material. Therefore, what follows is a description of the pattern of KCC2 immunoreactivity in the subiculum 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 also labeled (open arrow in Fig. 3E). KCC2 immunoreactivity was distributed in the cell body and in the proximal part of the dendrites. In patients with hippocampal sclerosis, KCC2-immunoreactive neurons were lost in parallel with the loss of neurons observed in Nissl-stained or Neu-N-immunostained sections of the stratum pyramidale in sclerotic regions of the CA1. However, some surviving neurons in sclerotic areas were also KCC2-immunoreactive.

Colocalization of NKCC and KCC2

In 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 (total number of NKCC-positive neurons examined = 251). In patients with hippocampal sclerosis, double-labeling experiments with anti-NKCC and anti-KCC2 antibodies revealed a high percentage of colocalization in nonsclerotic regions of CA4 (Figs. 3D–F) and subiculum, where most neurons (∼93%) coexpressed NKCC and KCC2 (total number of NKCC positive neurons examined = 224). However, the degree of colocalization of NKCC and KCC2 in neurons located in the subiculum/CA1 and in the sclerotic CA1, was lower (∼80%; total number of NKCC positive neurons examined = 181) than in nonsclerotic regions. Therefore, in these latter regions approximately 20% of the NKCC-immunoreactive neurons did not express KCC2. On the other hand, nearly all (>96%) KCC2-immunoreactive neurons coexpressed NKCC in all analyzed regions in patients with or without hippocampal sclerosis (Figs. 3A–F).

Parvalbumin immunostaining

Antibodies against PV characteristically stained nonpyramidal cell bodies and dense plexus formed by dendritic and axonal processes; the latter included the axon terminals of chandelier and basket cells (i.e., basket formations). Consistent with a previous study (Arellano et al., 2004) that used biopsy material obtained from epileptic patients with hippocampal sclerosis, we found alterations in inhibitory circuits that were prominent in the subiculum/CA1 region and in the sclerotic CA1 region (Figs. 3G–I and 4). These alterations were characterized by the presence of PV-immunoreactive basket formations that were distinctly denser and more complex than those seen in normal regions (Figs. 3G and 4A, D, G). The presence of these PV-immunoreactive hypertrophic basket formations suggests that some of the neurons in these regions were hyperinnervated by GABA-ergic interneurons. Double-labeling experiments were performed to determine whether those neurons hyperinnervated by PV-immunoreactive GABA-ergic terminals expressed NKCC or KCC2. In PV/NKCC double-labeled sections from the subiculum/CA1 and the sclerotic CA1 regions, 74% of the PV-immunoreactive hypertrophic basket formations (n = 61) innervated neurons expressing NKCC (Fig. 3I), whereas the remaining 26% innervated non-NKCC-expressing neurons.


Figure 4. Parvalbumin-immunoreactive basket formations innervating KCC2-immunoreactive neurons. The confocal images are from the same section and field taken from the subicum/CA1 regions of patients H48 (A–F) and H109 (G–I), illustrating the innervation patterns of KCC2-immunoreactive neurons (red) by PV-positive terminals (green). C, F, and I were obtained after combining images A and B, D and E, and G and H, respectively. Note that some PV-immunoreactive hypertrophic basket formations innervate KCC2-positive pyramidal cells (arrows in A–C). In D–I, arrows indicate hypertrophic PV-positive basket formations innervating the cell body of neurons devoid of KCC2. In G–I, open arrow point to a PV-immunoreactive interneuron that coexpressed KCC2. Images A–C represent stacks of 12 optical sections obtained at steps of 3.11 μm in the z axis. Images D–F represent stacks of 12 optical sections obtained at steps of 2.44 μm in the z axis (total 27 μm). Images G–I represent stacks of 16 optical sections obtained at steps of 3 μm in the z axis (total 45 μm). Scale bar: 33 μm for A–C and 23 μm for D–I.

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


  1. Top of page
  2. Abstract
  6. Acknowledgments

The present study demonstrates the expression of cation-chloride cotransporters (CCCs) KCC2 and putative NKCC1 in normal neocortex and epileptic temporal lobe of humans with hippocampal sclerosis. Western blot analysis revealed the presence of NKCC and KCC2 proteins in adult human temporal neocortex in all similar to those found in the brain of other vertebrates. Immunolabeling showed that these CCCs were located in the cell bodies and proximal dendrites of both pyramidal and nonpyramidal cells in the subiculum and in the hippocampus proper of patients with temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, NKCC is also expressed in glial cells while KCC2 is neuron specific. In the normal subiculum, subiculum/CA1, CA1, and CA4 regions of epileptic patients more than 95% of the pyramidal neurons coexpressed NKCC and KCC2. The degree of NKCC/KCC2 colocalization in the subiculum/CA1 and the sclerotic CA1 regions of patients with hippocampal sclerosis was lower (∼80%) than in the normal looking regions. Therefore, in the subiculum/CA1 region, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2.

PV immunolabeling confirmed the presence of abnormally dense (hypertrophic) basket formations surrounding the cell bodies and proximal dendrites of pyramidal cells in the subiculum/CA1 region of patients with hippocampal sclerosis (Arellano et al., 2004).

These PV-immunostained hypertrophic processes are axon terminals from GABA-ergic interneurons, mainly basket and chandelier cells. Their presence suggests that certain pyramidal neurons in the above areas become hyperinnervated by GABA-ergic axon terminals from interneurons, probably as a consequence of sprouting or relocation of axons from basket cells and other interneurons targeting pyramidal cells (Arellano et al., 2004). Double immunolabeling for PV and NKCC revealed that most hypertrophic basket formations (∼74%) innervated NKCC- immunostained pyramidal cells in the subiculum/CA1 and in the sclerotic CA1 regions. Double immunolabeling for PV and KCC2 showed that, in the same areas, 76% of the hypertrophic baskets innervated KCC2- immunostained neurons, whereas the remaining 24% innervated cells lacking KCC2.

NKCC and KCC2 proteins of adult human cerebral cortex

Western blot analysis revealed NKCC and KCC2 proteins in human temporal neocortex similar in molecular weight and glycosylation characteristics to those found in rat neocortex and hippocampus, as shown here and in previous 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 histopathological alterations and which showed normal electrical activity as characterized by ECoG. Thus, the CCC proteins identified by Western blot in this study most likely represent the normal characteristics of expression in the adult human cerebral neocortex. The NKCC antibody recognized a broad band of cotransporter immunoreactivity at about 170 kDa, consistent with the molecular mass of the glycosylated protein (Lytle et al., 1995; Payne et al., 1995; Haas and Forbush, 2000). This broad band could be resolved into two different components (∼165 and 175 kDa) by reducing film exposure. The NKCC monoclonal antibody used in the present study (T4) was generated against a fusion protein fragment encompassing the last 310 residues of the C-terminus of the human colonic NKCC (Lytle et al., 1995). The C-terminus displays more than 90% identity between NKCC isoforms (Payne et al., 1995), so the T4 antibody 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), we conclude that T4-immunoreactivity in the present material represents NKCC1 and that the doublet may correspond to the full length NKCC1 and a splice variant (see below). Using a polyclonal antibody directed against a 74-amino acid peptide located in the C-terminus of NKCC1 that does not recognize NKCC2, a protein doublet band (145–155 KDa) has also been detected in Westerns blots of rat forebrain (Plotkin et al., 1997a, 1997b). Similar NKCC1 protein doublets have been observed in Western blots from tissues other than brain. Moreover, the doublets appear using a variety of anti NKCC antibodies such as T4 (Xu et al., 1994; Liedtke et al., 2001), J3 (Flemmer et al., 2002) and the polyclonal one directed against a peptide comprising 74-amino acid residues of the C-terminus (Kaplan et al., 1996). The most common explanation offered in the literature is that the NKCC1 protein doublets represent different degrees of glycosylation of the same protein. Surprisingly, there are no published deglycolyation experiments to test this commonly held speculation. We found that following N-deglycosylation the doublet persists although the protein bands decreased in molecular mass. This suggests that the doublet in the present study represents two variants of NKCC1 because NKCC2 transcripts and proteins are not present in brain tissue. In mouse and humans there are at least two alternatively spliced RNA variants of NKCC1. In humans these variants have been named NKCCa and NKCC1b (Vibat et al., 2001). Both splice variants produce functional NKCC1s. NKCC1b is shorter than NKCC1a because it lacks 48 bp and is analogous to that described in mouse, which lacks exon 21 (Randall et al., 1997). Exon 21 encodes for a peptide of 16-amino acid residues. This peptide would give approximately 2-KDa difference between the two bands. Given the smearing typical of Western blots it is difficult to ascertain if the observed doublets in the present study specifically represent these NKCC1a and b variants. Clearly, this is an important issue that needs to be explored in future work.

We also demonstrate the presence in the human brain of KCC2, the neuronal-specific isoform of the K+-Cl cotransporter proteins (Payne et al., 1996). KCC2 isolated from human neocortex is an N-linked glycoprotein with a molecular weight of 140 kDa, similar in size to that from rat cortex as shown here and in previous reports (Williams et al., 1999). As expected, following treatment with N-glycosidase F the protein band migrates to 125 kDa, the size of the core protein (Payne et al., 1996).

NKCC and KCC2 are developmentally regulated proteins in rodents and humans (Plotkin et al., 1997a; Dzhala et al., 2005). Neuronal precursors and immature cortical neurons are depolarized by GABA (reviewed by Ben-Ari, 2002). This depolarization produces voltage-sensitive Ca2+ entry that plays a key role in neuronal differentiation, growth and maturation (Payne et al., 2003). The GABA-induced depolarizations ensue because the [Cl]i in immature neurons is higher than when in passive equilibrium due to an early expression of NKCC1 (Clayton et al., 1998). As development proceeds, KCC2 expression increases and GABA becomes hyperpolarizing, remaining like this in most mature central neurons (Plotkin et al., 1997a; Clayton et al., 1998; Rivera et al., 1999). The fate of NKCC in mature central neurons is controversial. Some claim that concomitant with the gradual appearance of KCC2, NKCC expression decreases reaching either very low levels (Plotkin et al., 1997a) or total disappearance in adult neurons (Yamada et al., 2004). Others show that NKCC expression appears early in development, increasing gradually until reaching a maximum that is maintained in adult brain (Clayton et al., 1998; Yan et al., 2001). Our results show that NKCC is definitely expressed in adult human neocortex.

Implications for Cl regulation and GABA-ergic function in normal and epileptogenic human cortex

We found that most cortical neurons coexpress NKCC1 and KCC2 suggesting that the level of intracellular Cl is determined by the functional interaction between these two cotransporters. The presence of a system that extrudes Cl (KCC2) coexisting with another that accumulates Cl (NKCC1) in the same cell suggests that Cl is tightly regulated (Gillen and Forbush, 1999). In nonneuronal cells NKCCs and KCCs are reciprocally regulated by negative feedback systems of kinases that are controlled 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 of Cl extrusion via KCC2 along with the concurrent active transport system that accumulates Cl in the same cells (NKCC) are likely to result in a higher than passive [Cl]i which means that the Cl equilibrium potential (ECl) will be much more positive than Em. This is expected because unlike in the case of skeletal muscle cells, the resting (passive) Cl permeability of neurons is relatively low (Alvarez-Leefmans, 1990). GABA signaling via A-type receptor channels in these cells will be depolarizing, due to Cl-efflux driven by the difference between Em and ECl. This hypothesis is supported by data from studies in animal models of epilepsy in which KCC2 mRNA and protein expression is down regulated. Under these conditions the impaired extrusion of Cl may leave NKCC1 unrestrained, leading to permanent positive shifts in ECl underlying and therefore GABAA depolarizing responses (Deisz, 2002; Rivera et al., 2002; Khalilov et al., 2003). In fact, it has been shown that a complete or partial disruption of KCC2 gene expression results in severe motor deficits, hippocampal hyperexcitability and frequent generalized seizures (Hubner et al., 2001; Woo et al., 2002; Rivera et al., 2004). In addition, it has been shown that Cl accumulation and GABA-induced depolarizations are abolished in NKCC1 knockout mice (Sung et al., 2000) and the inhibition of NKCC blocks spontaneous epileptiform activity (Hochman et al., 1995, 1999; Schwartzkroin et al., 1998; Hochman and Schwartzkroin, 2000; Fukuda, 2005). Thus, we hypothesize that, in temporal lobe epileptic patients with hippocampal sclerosis, the lack of KCC2 in NKCC1-expressing cells may contribute to the depolarizing responses induced by GABA-ergic signaling through GABAA receptors in neurons within the subiculum and its transitional region with CA1 as reported previously (Cohen et al., 2002). These cells discharge interictal-like bursts and presumably act as pacemakers in generating interictal synchrony (Cohen et al., 2002, 2003). Remodeling of GABA-ergic circuits in the subiculum/CA1 and sclerotic CA1 regions in epileptic patients include the hyperinnervation of some pyramidal cells by PV-positive GABA-ergic basket formations (Arellano et al., 2004). We found that around 25% of PV-positive hypertrophic basket formations innervated pyramidal cells lacking KCC2, whereas most of hyperinnervated pyramidal cells expressed putative NKCC1. Thus, it is likely that some hypertrophic GABA-ergic basket formations innervate pyramidal cells that express NKCC1 but lack KCC2. In conclusion, the findings described in the present study demonstrate: (1) the presence of KCC2 and putative NKCC1 proteins in adult human brain in all similar to those found in rat cerebral cortex; (2) that in patients with hippocampal sclerosis, 20% of NKCC-immunoreactive neurons in the subiculum/CA1 do not express KCC2. In the same region, approximately 75% of hypertrophic GABA-ergic terminals innervate NKCC-positive neurons whereas 25% innervate KCC2-negative cells. Altogether these findings represent a morphological substrate that could explain the anomalous GABA depolarizing responses found in pyramidal cells by Cohen et al. (2002). These responses participate in the generation of seizure activity in the human epileptogenic sclerotic hippocampus. Consistent with our results and conclusions are recent observations (Dzhala et al., 2005) showing that epileptiform activity in perinatal humans and rodents is correlated to relatively high expression of NKCC1 with respect to KCC2.


  1. Top of page
  2. Abstract
  6. Acknowledgments

Acknowledgments:  This work was supported by the Ministerio de Ciencia y Tecnología grants BFI 2003-01018 and BFU2006-03855 to A.M. and BFI 2003-02745 and BFU2006-13395 to J. de F., and by the NINDS-NIH grant NS29227 to 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. Payne for providing the KCC2 antisera.


  1. Top of page
  2. Abstract
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