Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy

Abstract

Changes in chloride (Cl-) homeostasis may be involved in the generation of some epileptic activities. In this study, we asked whether Cl- homeostasis, and thus GABAergic signaling, is altered in tissue from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Slices prepared from this human tissue generated a spontaneous interictal-like activity that was initiated in the subiculum. Records from a minority of subicular pyramidal cells revealed depolarizing GABA(A) receptor-mediated postsynaptic events, indicating a perturbed Cl- homeostasis. We assessed possible contributions of changes in expression of the potassium-chloride cotransporter KCC2. Double in situ hybridization showed that mRNA for KCC2 was absent from approximately 30% of CaMKIIalpha (calcium/calmodulin-dependent protein kinase IIalpha)-positive subicular pyramidal cells. Combining intracellular recordings with biocytin-filled electrodes and KCC2 immunochemistry, we observed that all cells that were hyperpolarized during interictal events were immunopositive for KCC2, whereas the majority of depolarized cells were immunonegative. Bumetanide, at doses that selectively block the chloride-importing potassium-sodium-chloride cotransporter NKCC1, produced a hyperpolarizing shift in GABA(A) reversal potentials and suppressed interictal activity. Changes in Cl- transporter expression thus contribute to human epileptiform activity, and molecules acting on these transporters may be useful antiepileptic drugs.

Figure 1.

Two distinct pyramidal cell behaviors during interictal events. A, A subicular pyramidal cell hyperpolarized (top trace) during an extracellular epileptiform burst (bottom trace). B, A pyramidal cell (top) depolarized and discharged during epileptiform events (bottom). Action potentials are cut. Photomicrographs of each biocytin-filled cell are shown on the right. Scale bars, 25 μm.

Figure 2.

IPSP reversal potential (V_rev) for cells of the epileptogenic area. V_rev was estimated from plots of the amplitude of synaptic events, evoked by focal stimulation in 10 μm NBQX and 50 μm d,l-APV, against membrane potential. A, Pyramidal cells with values for V_rev of −75 mV (left) and −48 mV (right). Top, Synaptic events at different potentials (V_m). Bottom, IPSP amplitude plotted against membrane potential. stim., Stimulation; rec., recording. B, Distribution of V_rev for 30 subicular pyramidal cells. C, Correlation of V_rev and resting potential (V_rest) for 30 cells. Reversal potentials were depolarizing with respect to rest in six cells, above the diagonal line, and hyperpolarizing in 24 cells below the line.

Figure 3.

KCC2 mRNA is absent from a minority of subicular neurons. A, KCC2 mRNA expression detected by in situ hybridization in a section from human temporal lobe. B, Hybridization with sense mRNA revealed no nonspecific signal. C–F, Double in situ hybridization for KCC2 mRNA (BM purple) and CaMKIIα (S³⁵) in dentate gyrus (C), CA2 (D), subiculum proximal to CA1 (D), and distal subiculum (E, F). Sections correspond to the labeled squares in A. Arrows indicate CaMKII-positive cells with very low KCC2 mRNA levels. G, Higher magnification of the area inside the black square in F. H, Percentage of CaMKIIα mRNA-positive cells that express KCC2 mRNA in dentate gyrus (DG), CA2, and proximal (Prox) and distal (Dis) zones of the subiculum (Sub) plotted from Table 1. Error bars indicate SDs. Scale bars: A, B, 3 mm; C–F, 50 μm; G, 15 μm.

Figure 4.

Immunodetection of the Cl⁻ cotransporter KCC2. A, KCC2 immunostaining of subiculum (Sub), with asterisks indicating positive pyramidal cells. B, KCC2 immunostaining of dentate gyrus (DG) showing the molecular layer (M), granule cell layer (GCL), and hilus (H). Inset, Higher magnification of GCL. C, Subicular KCC2-positive pyramidal cells (PC) and interneuron (IN). D, KCC2-negative (PC−) and positive (PC+) subicular pyramidal cells. Scale bars: A, B (inset), C, D, 25 μm; B, 50 μm.

Figure 5.

Correlation of pyramidal cell behavior during epileptiform events with KCC2 immunostaining. A–C, Left panels are intracellular (top) and extracellular (bottom) records. Right panels are images of a recorded cell filled with biocytin (red) and immunostained for KCC2 (green). Biocytin-filled, KCC2-positive cells are yellow in the merge. Red arrowheads indicate the soma, and open arrowheads indicate the apical dendrite. Confocal fluorescent images of 5–10 μm stack thickness are shown. A, Six of six hyperpolarized cells were immmunopositive for KCC2. B, Four of seven cells depolarized during interictal bursts were negative for KCC2. C, Three of seven cells depolarized during interictal bursts were positive for KCC2. Some cytoplasmic signal may be nonspecific lipofuscin staining (Yin, 1996). D, Correlation of IPSP reversal potential with the presence (yellow) or absence (red) of KCC2 (n = 13). Depolarizing GABAergic events are positive, and hyperpolarizing events are negative.

Figure 6.

Effects of bumetanide on GABAergic signaling and interictal activity. A, Bumetanide (8 μm) hyperpolarized the reversal potential of GABAergic IPSPs. Top traces show synaptic events at different membrane potentials (V_m). Reversal potentials are indicated with an arrow. Bottom traces plot IPSP amplitude against membrane potential. B, Bumetanide blocked spontaneous epileptiform bursts. Top traces show that extracellular interictal-like field potentials (left) are blocked by bumetanide (right). The bottom plot shows reversible changes in the instantaneous frequency of interictal-like discharges induced by bumetanide application and washout. C, Bumetanide does not affect epileptiform bursts induced by increasing extracellular K⁺ to 12 mm in the presence of 100 μm picrotoxin (PTX).