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. 2015 May;138(Pt 5):1208-22.
doi: 10.1093/brain/awv067. Epub 2015 Mar 12.

Astrocyte Uncoupling as a Cause of Human Temporal Lobe Epilepsy

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Free PMC article

Astrocyte Uncoupling as a Cause of Human Temporal Lobe Epilepsy

Peter Bedner et al. Brain. .
Free PMC article

Abstract

Glial cells are now recognized as active communication partners in the central nervous system, and this new perspective has rekindled the question of their role in pathology. In the present study we analysed functional properties of astrocytes in hippocampal specimens from patients with mesial temporal lobe epilepsy without (n = 44) and with sclerosis (n = 75) combining patch clamp recording, K(+) concentration analysis, electroencephalography/video-monitoring, and fate mapping analysis. We found that the hippocampus of patients with mesial temporal lobe epilepsy with sclerosis is completely devoid of bona fide astrocytes and gap junction coupling, whereas coupled astrocytes were abundantly present in non-sclerotic specimens. To decide whether these glial changes represent cause or effect of mesial temporal lobe epilepsy with sclerosis, we developed a mouse model that reproduced key features of human mesial temporal lobe epilepsy with sclerosis. In this model, uncoupling impaired K(+) buffering and temporally preceded apoptotic neuronal death and the generation of spontaneous seizures. Uncoupling was induced through intraperitoneal injection of lipopolysaccharide, prevented in Toll-like receptor4 knockout mice and reproduced in situ through acute cytokine or lipopolysaccharide incubation. Fate mapping confirmed that in the course of mesial temporal lobe epilepsy with sclerosis, astrocytes acquire an atypical functional phenotype and lose coupling. These data suggest that astrocyte dysfunction might be a prime cause of mesial temporal lobe epilepsy with sclerosis and identify novel targets for anti-epileptogenic therapeutic intervention.

Keywords: gap junction coupling; gap junction protein alpha 1; hippocampal sclerosis; inflammation; temporal lobe epilepsy.

Figures

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Increasing evidence suggests glial cell involvement in CNS disorders. Using techniques including patch clamp recording, EEG/video monitoring and fate mapping in human hippocampal tissue and in a mouse model, Bedner et al. reveal a causal role for astrocyte dysfunction in the aetiology of mesial temporal lobe epilepsy with sclerosis.
Figure 1
Figure 1
Characterization of astrocytes and NG2 cells in non-sclerotic and sclerotic human hippocampus. (A) The whole-cell current pattern of an astrocyte (left; 50 ms voltage steps ranging from −160 to +20 mV; 10 mV increments; Vhold = −80 mV) was dominated by a passive resting conductance. Rapid application of glutamate to an outside-out patch failed to induce outward currents at positive voltages (middle and right), indicating the absence of ionotropic receptors. The inward currents were due to glutamate uptake. (B) Gap junction coupling between hippocampal astrocytes visualized by diffusion of biocytin from a single cell, filled with the tracer through the patch pipette during a 20 min whole-cell recording. Scale bar = 100 µm. (C) Typical whole-cell current pattern of an NG2 cell (left). De- and hyperpolarization activated time- and voltage-dependent out- and inward currents. Flash photolysis of caged glutamate activated currents with a linear current/voltage plot (middle and right), indicating the expression of ionotropic receptors. (D) Biocytin filling of NG2 cells revealed lack of tracer coupling. Scale bar = 25 µm. (E) Complex current pattern resembling an NG2 cell in human hippocampal sclerosis (left). Application of glutamate to an outside-out patch excised from the soma demonstrates expression of ionotropic glutamate receptors with a linear current/voltage curve (middle and right). (F) Intercellular diffusion of biocytin was never observed in hippocampal sclerosis. Scale bar = 25 µm. (G) Some of these cells co-expressed ionotropic glutamate receptors and glutamate transporters, as revealed by the incomplete block of inward currents by NBQX. (H) Almost complete loss of cells with passive current pattern was noted in the hippocampus of patients with MTLE-HS. Number of investigated cells in brackets; c.p. = current pattern.
Figure 2
Figure 2
Loss of bona fide astrocytes and gap junction coupling in the hippocampus of kainate-injected mice. (A) Representative example showing abolished tracer coupling in sclerotic slices obtained from epileptic mice 3 mpi (top left). In contrast, in the contralateral hippocampus astrocytes displayed abundant gap junction coupling, resembling control conditions (bottom left). Scale bar = 100 µm. Whole-cell currents of the filled cells were elicited as described in the legend to Fig. 1A (right). (B) Summary of tracer coupling experiments. The extent of intercellular biocytin diffusion was compared in sclerotic and non-sclerotic slices of the injected hemisphere, and in slices from the contralateral hippocampus. Three and 6 mpi, no spread of biocytin could be detected in sclerotic slices (3 months: n = 12 biocytin-filled passive cells from six animals; 6 months: n = 6 filled passive cells, three animals). Astrocytes were still coupled in non-sclerotic hippocampal slices of the injected hemisphere (3 months: 30 ± 20.1 coupled cells, n = 11 slices, six animals; 6 months: 27.5 ± 17.8 coupled cells, n = 10 slices, five animals) and slices obtained from the contralateral hippocampus (3 months: 40.6 ± 22.5 coupled cells, n = 10 slices, six animals; 6 months: 44.9 ± 27.7 coupled cells, n =11 slices, three animals). Nine months after status epilepticus, complete loss of cells with passive current pattern was observed in sclerotic segments of the hippocampus (n = 18 screened slices, six animals) whereas in non-sclerotic hippocampal slices ipsilateral to the injection, and in the contralateral hippocampus, astrocytes coupled to 23.8 ± 16.8 (n = 13 slices, five animals) and 29.6 ± 21 (n = 18 slices, five animals) cells, respectively. Gap junction coupling in non-sclerotic ipsilateral slices did not differ from the contralateral side. (C) Loss of SR101 uptake by astrocytes in the sclerotic hippocampus. Incubation with SR101 of slices from epileptic mice 9 mpi resulted in astrocytic labelling in the contralateral CA1 region. In the ipsilateral hippocampus, no labelled cells were detected (n = 3 slices, three animals). Scale bar = 15 µm.
Figure 3
Figure 3
Decreased tracer coupling and impaired K+ clearance in the latent period after kainate injection. (A) Representative example showing reduced tracer coupling in the ipsilateral hippocampus 4 hpi. Scale bar = 100 µm. Insets show current responses of the filled cells (stimulus protocol as in Fig. 1A; horizontal bar, 10 ms; vertical bar, 2.5 nA). (B) Ipsilateral TUNEL performed 4 and 6 hpi. No TUNEL-positive cells could be detected in the pyramidal layer 4 hpi (n = 7 slices from three animals), whereas abundant staining of pyramidal neurons was found underneath the injection site 6 hpi (n = 7 slices from three animals). sr = stratum radiatum; sp = stratum pyramidale; so = stratum oriens. Scale bar = 250 µm. (C) Summary of astrocytic gap junction coupling in dorsal slices of the ipsilateral hippocampus, expressed as percentage of the number on the contralateral side. Ipsi- and contralateral measurements were always conducted in the same slice. Mice, which had experienced seizures, were excluded from the study. In the ipsilateral hippocampus, astrocytic gap junction coupling was significantly reduced at each time point investigated (4 hpi: 55.2 ± 31.2 versus 119 ± 47.6 coupled cells, P = 0.01; n = 12 slices from eight animals; 1 day post injection: 53.8 ± 11.7 versus 102.6 ± 21.8 coupled cells, P = 0.004; n = 10 slices from five animals; 4–5 days post injection: 28 ± 18.2 versus 79.6 ± 27.9 coupled cells, P = 0.001; n = 18 slices from eight animals). Sham injection did not alter gap junction coupling (99.9 ± 37.6 versus 96.7 ± 30.7 coupled cells, P = 0.6; n = 11 slices from six animals). (D, left) To investigate the dependence of K+ clearance on gap junction coupling, astrocytes were patched and held in the current clamp mode. [K+]o increases were induced by Schaffer collateral stimulation (dashed lines). Cells were visualized by two-photon excitation fluorescence microscopy (middle and right panel, patch pipette indicated by dotted lines; Scale bar = 50 µm). Note the prominent dye coupling (Alexa Fluor® 594) to neighbouring astrocytes in control (middle) and reduced gap junction coupling ipsilaterally (right). (E) Quantification of dye coupling (contralateral, 18.1 ± 12.8 coupled cells; ipsilateral, 8 ± 4.8 coupled cells, P = 0.032, n = 9 and 10 slices, five and six animals, respectively). (F and G) The long-lasting, largely K+-dependent component of astrocyte voltage responses to stimulation (ΔVK, dotted lines) was analysed without (blue and red traces) and with (grey traces) the gap junction blocker carbenoxolone (CBX, 50 µM) present, and normalized to the fibre volley (arrowhead, synaptic transmission blocked, stimulus artefacts removed for clarity). On the contralateral side, responses were significantly larger in carbenoxolone-treated compared to untreated slices (artificial CSF, 0.187 ± 0.091, n = 14 cells, seven animals; artificial CSF + carbenoxolone, 0.298 ± 0.09, n = 11 cells, seven animals, P = 0.006) whereas carbenoxolone had no effect on ΔVK in ipsilateral slices (artificial CSF, 0.193 ± 0.068, n = 10 cells, six animals; artificial CSF + carbenoxolone, 0.197 ± 0.055, n = 12 cells, seven animals, P = 0.887). The ipsi- and contralateral carbenoxolone values also differed significantly (P = 0.003). FV = fibre volley; *significantly different from the contralateral side (t-test); #significantly different from sham (ANOVA and Tukey test).
Figure 4
Figure 4
In the course of epilepsy astrocytes acquire an abnormal phenotype. (A) Schematic of fate mapping experiments. Activation of EYFP expression in GJA1-positive glial cells was induced by intraperitoneal injection of tamoxifen. Four weeks later, kainate was unilaterally injected into the cortex. Fluorescent cells were analysed electrophysiologically and immunohistochemically 5, 90 and 180 days after kainate injection. (B) Representative example of an EYFP-positive cell lacking gap junction coupling and showing abnormal input resistance (43 MΩ), distinct from bona fide astrocytes. Scale bar = 20 µm. (C) Tracer coupling analysis of EYFP-positive cells at different time points after kainate injection shows significant reduction of gap junction coupling already during the latent period (68.6 ± 33.9 versus 131.4 ± 33 coupled cells, P = 0.009, n = 27 slices from six animals), and complete loss of gap junction coupling after 6 months (n = 22 slices from five animals). (D) The proportion of EYFP-positive cells with membrane currents atypical for astrocytes increased with time after kainate injection (5 days post injection: n = 30 slices from six animals; 3 mpi: n = 18 slices from four animals; 6 mpi: n = 24 slices from six animals). (E) TUNEL/GFAP/Draq5 triple staining of coronal brain slices at 5 days and 3 months after kainate injection. No apoptotic astrocytes could be detected in sclerotic and non-sclerotic parts of ipsilateral hippocampi. sr = stratum radiatum; sp = stratum pyramidale; so = stratum oriens. Scale bar = 25 µm.
Figure 5
Figure 5
Pro-inflammatory cytokines and lipopolysaccharide inhibit astrocytic gap junction coupling. (A) Kainate injection did not affect astrocytic gap junction coupling in TLR4 knockout mice at 1 day post injection (121.2 ± 35.6 versus 115.9 ± 50.2 coupled cells, P = 0.87, n = 10 slices from five animals). Data were normalized to the contralateral side. Ipsi- and contralateral gap junction coupling were always compared in the same brain slices. (B) Tracer coupling analysis was performed in situ in acute brain slices 3–4.5 h after incubation with IL1B, IL1B/TNF (10 ng/ml), or lipopolysaccharide (LPS, 1 µg/ml). The cytokines and lipopolysaccharide significantly decreased gap junction coupling (control: 87.8 ± 18.2 coupled cells, n = 33 slices from 15 animals; IL1B: 60.5 ± 9.5 coupled cells, P = 0.002, n = 10 slices from seven animals; IL1B + TNF: 53.5 ± 15.6 coupled cells, P < 0.0001, n = 15 slices from 11 animals; lipopolysaccharide: 47.9 ± 16.8 coupled cells, P = 0.0016, n = 14 slices from four animals). The effect of the cytokines on gap junction coupling was prevented by addition of 100 µM dibutyryl cyclic AMP (IL1B + TNF + dibutyryl cyclic AMP: 88.2 ± 42.1 coupled cells, P = 0.93, n = 18 slices from six animals). Data were normalized to gap junction coupling in control slices (vehicle incubation). (C) In vivo effect of lipopolysaccharide on astrocytic gap junction coupling. Gap junction coupling was assessed in acute slices 5 days after i.p. injection of lipopolysaccharide (5 mg/kg). Lipopolysaccharide significantly decreased gap junction coupling (56.5 ± 27.5 coupled cells, n = 21 slices from five animals versus 104 ± 19 coupled cells, n = 18 slices from five animals, P = 0.01). Treatment of lipopolysaccharide-injected animals for 5 days with levetiracetam (150 mg/kg, i.p. two injections daily) fully restored gap junction coupling (139.3 ± 19.9 coupled cells, P = 0.369, n = 19 slices from five animals). Data were normalized to control mice (vehicle injection). *Significantly different (ANOVA and Tukey test); db-cAMP = dibutyryl cyclic AMP.

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