Retarded kindling progression in mice deficient in the extracellular matrix glycoprotein tenascin-R

Authors

  • Katrin Hoffmann,

    1. Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany
    Search for more papers by this author
  • Elena Sivukhina,

    1. Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
    Search for more papers by this author
  • Heidrun Potschka,

    1. Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany
    Search for more papers by this author
  • Melitta Schachner,

    1. Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
    2. Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey, U.S.A.
    Search for more papers by this author
  • Wolfgang Löscher,

    1. Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany
    Search for more papers by this author
  • Alexander Dityatev

    1. Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
    2. Institut für Neurophysiologie und Pathophysiologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
    3. Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova, Italy
    Search for more papers by this author

Address correspondence to Melitta Schachner, Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Falkenried 94, D-20251 Hamburg, Germany. E-mail: melitta.schachner@zmnh.uni-hamburg.de; or to Alexander Dityatev, Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, 16163 Genova, Italy. E-mail: alexander.dityatev@iit.it

Summary

Purpose: We investigated the role of the extracellular matrix glycoprotein tenascin-R (TNR) in formation of a hyperexcitable network in the kindling model of epilepsy. The idea that TNR may be important for this process was suggested by previous studies showing that deficiency in TNR leads to abnormalities in synaptic plasticity, perisomatic GABAergic inhibition and more astrocytes in the hippocampus of adult mice.

Methods: Constitutively TNR deficient (TNR−/−) mice and their wild-type littermates received repeated electrical stimulation in the amygdala over several days until they developed fully kindled generalized seizures at which time their brains were studied immunohistochemically.

Results: In TNR−/− mice, kindling progression was retarded compared with wild-type littermate controls. Morphological analysis of the mice used for the kindling studies revealed that, independently of genotype, numbers of parvalbumin-positive interneurons in the dentate gyrus correlated positively with afterdischarge threshold alterations in kindled mice. The kindling-induced increase in the number of S100 expressing astrocytes in the dentate gyrus was enhanced by TNR deficiency and correlated negatively with the kindling rate.

Discussion: Our data support the view that TNR promotes formation of a hyperexcitable network during kindling and suggest that an increase in S100-expressing astrocytes may contribute to retarded epileptogenesis in TNR−/− mice.

Epilepsy is one of the most common neurological disorders, but the pathophysiologic processes leading to epilepsy remain poorly understood. Here we addressed the question whether the extracellular matrix glycoprotein tenascin-R (TNR), previously shown to be important for synaptic plasticity and perisomatic inhibition in the hippocampus (Saghatelyan et al., 2000; Bukalo et al., 2001; Saghatelyan et al., 2001; Bukalo et al., 2007), may affect epileptogenesis. TNR is expressed exclusively in the central nervous system (CNS) of vertebrates (Pesheva et  al., 1989; Dityatev & Schachner, 2003; Pesheva et al., 2006). TNR is synthesized and secreted by oligodendrocytes, Purkinje cells, motoneurons, and subpopulations of interneurons (Bartsch et al., 1993; Fuss et al., 1993; Wintergerst et al., 1993; Woodworth et al., 2002; Becker et al., 2003). In contrast to oligodendrocytes, TNR expression in neurons does not decrease after the postnatal period (Morganti et al., 1990), and the secreted protein is concentrated in perineuronal nets (Brückner et al., 2000; Dityatev et al., 2007).

Constitutive TNR deficiency in mice causes several abnormalities in synaptic transmission and plasticity in the CA1 region of the hippocampus, including reduction in N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and increase in basal excitatory synaptic transmission in CA3-CA1 synapses (Saghatelyan et al., 2001). Also, hippocampal γ-oscillations, cortical electroencephalography (EEG) activity, and cortical and hippocampal auditory-evoked potentials are abnormally enhanced in TNR deficient (TNR−/−) mice (Gurevicius et al., 2004). The increase in excitatory transmission is accompanied by reduction of perisomatic GABAergic inhibition of CA1 pyramidal cells (Saghatelyan et al., 2000; Bukalo et al., 2001; Saghatelyan et al., 2001; Nikonenko et al., 2003). In the dentate gyrus, however, the ratio of inhibitory to excitatory cells is higher and the perisomatic GABAergic input to granule cells is increased in TNR−/− mice when compared to wild-type control littermates (TNR+/+ mice) (Morellini, Sivukhina, Stoenica,  Oulianova, Bukalo, Dityatev, Irintchev, Schachner M, unpublished). In vitro analysis of population spikes in hippocampal slices of TNR−/− mice revealed a significant increase in multiple spikes in the CA1 region in response to 1 Hz stimulation, as compared with TNR+/+ mice (Brenneke et al., 2004a). Adult TNR−/− mice also have high numbers of glial fibrillary acidic protein (GFAP)-positive astrocytes in the CA1 and CA3 regions (Brenneke et al., 2004a). To test whether these abnormalities alter the susceptibility to develop seizures in TNR−/− mice, we had used the pilocarpine model of epilepsy. No genotype-specific differences were found with regard to the time-course of pilocarpine-induced status epilepticus as well as spontaneous seizures that developed following the latency period (Brenneke et al., 2004a). However, there were striking interindividual differences in pilocarpine-elicited responses (from no seizures to death) in TNR−/− and TNR+/+ mice in these experiments, which are quite common for the pilocarpine model and might have masked TNR-related features of epileptogenesis. We therefore decided to reinvestigate the susceptibility to seizures in TNR−/− mice using the amygdala kindling model. Kindling is widely accepted as a functional model of temporal lobe epilepsy in which the altered neuronal response develops in the absence of gross morphological damage, such as that seen in many other epilepsy models (e.g., the pilocarpine and kainate models) (Morimoto et al., 2004). In the kindling model, repeated electrical stimulation (with initially subconvulsive currents) of the amygdala, hippocampus, or other regions of the limbic system results in the development of focal (limbic) seizures strikingly similar to those of complex partial seizures of temporal origin in humans. With ongoing stimulations, secondary generalized (clonic) seizures develop in addition to focal seizures. In the fully kindled state with constant generalized seizures, the enhanced sensitivity to electrical stimulation seems to be permanent. One of the most dramatic changes that occur during kindling is the increased propagation of the epileptic discharge or evoked electrographic measure (afterdischarge) from the site of stimulation (e.g., the amygdala) to other sites and the recruitment of those sites into the discharge.

Here, we compared kindling induced epileptogenesis in TNR+/+ and TNR−/− mice based on behavioral seizure activity and EEG recordings. Furthermore, we attempted to find cellular correlates underlying the genotype-related and interindividual variability in kindling susceptibility.

Materials and Methods

Animals

TNR-deficient (TNR−/−) mice and wild-type (TNR+/+) male littermates used in the present study were offspring of heterozygous mice with a C57BL/6J genetic background after five backcrosses of mice from the original C57Bl/6J × 129/Ola colony (Weber et al., 1999). The animals were bred at the specific pathogen-free animal facility of the University Medical Center Hamburg-Eppendorf. The genotype of each animal was identified using polymerase chain reaction (PCR) of genomic DNA isolated from tail biopsies before the experiments.

Ten 4- to 6-month-old TNR−/− animals weighing 20–25 g and 10 TNR+/+ littermates of the same age, with body weight of 22–24 g, were successfully implanted with the stimulation-recording electrode targeted to the amygdala. Of these, eight TNR+/+ mice and seven TNR−/− mice were successfully kindled. Kindling was done with two cohorts of mice in two consecutive sets of experiments. The two cohorts of mice did not differ in kindling parameters and were therefore combined for final analyses. Since we detected differences in kindling between the genotypes in the first set of experiments, the second cohort of mice was subjected to morphological analysis. These included three kindled TNR−/− mice, three nonkindled TNR−/− mice, four kindled TNR+/+ mice, and two nonkindled TNR+/+ mice. The nonkindled mice were implanted with electrodes that were not used for stimulation and recording, and we therefore refer to these in the text as sham controls. Additionally, eight naive 5-month-old TNR−/− mice and eight TNR+/+ littermates were used for the morphological analyses.

Mice were housed under controlled conditions (ambient temperature 24°–25°C, humidity 50%–60%, lights on from 6:00 a.m. to 6:00 p.m.). All efforts were made to minimize both the suffering (see below) and the number of animals used. The procedures used in this study were conducted in accordance with the national and international guidelines on the ethical use of animals..

Surgery and kindling

Mice were anesthetized with chloralhydrate [500 mg/kg, intraperitoneally (i.p.)], and a stimulation-recording bipolar electrode was stereotaxically implanted in the right amygdala using the following coordinates with bregma as the reference according to the atlas of Paxinos and Franklin (2001): −1.6 mm rostrocaudal, −3.4 mm lateral, and −4.7 mm dorsoventral. Only animals with a correct location of the stimulation-recording electrode within the amygdala were used for evaluation of data. A stainless-steel screw electrode of 1.6 mm diameter was placed above the left parietal cortex and served as indifferent reference electrode. Electrodes were made of 0.1 mm Teflon-insulated stainless steel wire soldered with a microelectronic connector. The electrode assembly was combined to form a female connector and was anchored to the skull with dental acrylic cement. After surgery, animals received 0.1 mg/kg buprenorphine for postoperative analgesia and were allowed to recover for a period of 1 week. Analgesia was done postoperatively to reduce the risk of apnea, as buprenorphine exhibits strong depressive effects on respiration in mice especially in combination with  chloralhydrate.

The mice were then electrically kindled via the  electrode in the amygdala. The threshold for inducing afterdischarges was determined prior and after the kindling process by an ascending stair-step procedure, starting at an initial current intensity of 25 μA (1 s train, 1 ms stimulus at 50 Hz) followed by increases in current intensity by about 20% of the previous current at intervals of 1 min until the stimulation-induced afterdischarges of at least 3 s duration. For kindling, the animals were stimulated once daily with a current 20% higher than the individual initial (prekindling) afterdischarge threshold (ADT) until three stage 5 seizures were elicited. An animal was considered “fully kindled” after the first stage 5 seizure. Severity of seizures was classified according to Racine (1972): Stage 1, immobility and facial automatisms; stage 2, head nodding, associated with more severe facial clonus; stage 3, unilateral forelimb clonus; stage 4, rearing and bilateral forelimb clonus; stage 5, rearing and falling accompanied by generalized tonic–clonic seizures. In addition to seizure severity, seizure duration and afterdischarge duration were determined. Seizure duration was the period of limbic and/or motor seizures. Afterdischarge duration was the total time of spikes (defined as discharges at least 2-fold higher than baseline noise) in the EEG, including time of stimulation.

Preparation of tissue samples and immuno histochemistry

After completion of kindling and a subsequent stimulation-free period of approximately 4 weeks, the persistence of the fully kindled state was proven by an additional stimulation at the same intensity used in the kindling procedure, which resulted in fully-kindled seizures. Four days after stimulation, the animals were deeply anesthetized with chloralhydrate and perfused with 0.9% saline for 60 s followed by 4% formaldehyde in 0.1 M phosphate buffer by cardiac puncture via the left ventricle. Tissue preparation and immunohistochemical stainings were performed as described previously (Irintchev et al., 2005). The brains were dissected and postfixed overnight at 4°C in the fixative used for perfusion followed by immersion into 0.1 M phosphate buffer, pH 7.3, containing 15% sucrose solution for an additional day at 4°C. The tissue was then frozen for 2 min in 2-methyl-butane (isopentane) precooled to −30°C in the cryostat, and stored in liquid nitrogen until sectioned. Serial coronal sections of 25 μm thickness were cut in a caudal-to-rostral direction on a Leica CM3050 cryostat (Leica Instruments, Nußloch, Germany). Sections from 1 mm tissue thickness were collected on a series of 10 SuperFrost Plus glass slides (Roth, Karlsruhe, Germany) so that four sections 250 μm apart were present on each slide. The sections were air-dried for at least 1 h at room temperature and stored in boxes at −20°C until stainings were performed.

Prior to the immunofluorescence staining, antigen demasking using 0.01 M sodium citrate solution, pH 9.0, was done in a water bath at 80°C for 30 min (Jiao et  al., 1999). Blocking of nonspecific binding sites was performed for 1 h at room temperature using phosphate-buffered saline (PBS), pH 7.3, containing 0.2% Triton X-100 (Roche Diagnostics, Mannheim, Germany), 0.02% sodium azide, and 5% normal serum from the species in which the secondary antibody was produced. Incubation with the primary antibody, anti-parvalbumin (PV; mouse monoclonal, clone PARV-19; Sigma, Taufkirchen, Germany) or anti-S100 (rabbit polyclonal; DakoCytomation, Göttingen, Germany) diluted 1:1000 or 1:500, respectively, in PBS containing 0.5%λ-Carrageenan (Sigma, Taufkirchen, Germany) and 0.02% sodium azide, was carried out at 4°C for 3 days. This anti-S100 antibody labeled in mice only astrocytes and not neurons in the procedure described (Irintchev et al., 2005). After washing in PBS (three times for 15 min each wash at room temperature), the appropriate secondary antibody conjugated with Cy3 (Jackson ImmunoResearch  Laboratories, Dianova,  Hamburg, Germany) diluted 1:200 in PBS-Carrageenan solution was applied for 2 h at room temperature. Finally, after a subsequent wash in PBS, cell nuclei were stained for 10 min at room temperature with bis-benzimide solution (Hoechst 33258 dye, 5 μg/ml in PBS; Sigma, Taufkirchen, Germany), and sections were mounted under coverslips with antifading medium (Fluoromount G; Southern Biotechnology Associates, Biozol, Eching, Germany).

Stereological analysis of parvalbumin- and S100-positive cells

Numerical densities were estimated using the optical disector method as previously described (Irintchev et al., 2005; Nikonenko et al., 2006). Counting was performed on an Axioskop microscope (Zeiss, Oberkochen,  Germany) equipped with a motorized stage and Neurolucida software-controlled computer system  (MicroBrightField Europe, Magdeburg, Germany). The volume of the dentate gyrus (DG) was estimated using spaced-serial sections (250 μm interval) and the Cavalieri principle (Nikonenko et al., 2006). The borders of the DG were defined by the nuclear staining pattern (Plan-Neofluar, Zeiss MicroImaging, Munich Germany; 10×/0.3 objective) according to criteria described by Long and colleagues (1998). The numerical density of PV-positive interneurons as well as S100-positive astrocytes was estimated by counting nuclei of immunolabeled cells within systematically randomly spaced optical disectors in the DG. The parameters for this analysis were: Guard space depth 2 μm, base and height of the disector 3,600 μm2 and 10 μm, respectively, distance between the optical disectors 60 μm, objective 40× Plan-Neofluar 40×/0.75. Left and right hippocampal areas were evaluated in four sections each. All results shown are averaged bilateral values. The counts were performed on coded preparations by one observer.

Statistical analysis

All numerical data are presented as group mean values with standard error of the mean (SEM). Parametric and nonparametric tests, indicated in the text and figure legends, were used to analyze the data as appropriate. The accepted level of significance was 5%. The nonparametric Spearman's test was used to analyze correlations between morphological and electrophysiological data sets.

Results

Development of kindling in TNR deficient mice

The prekindling (initial) ADT did not significantly differ between TNR+/+ and TNR−/− mice (Fig. 1). Mice of both genotypes displayed a remarkable variation in initial ADT with a minimum current of 42 μA and a maximum current of 700 μA in TNR−/− mice compared, respectively, with 60 μA and 500 μA for TNR+/+ mice. Following the kindling process, the postkindling ADTs were decreased in comparison to the prekindling ADTs [p = 0.035, two-way analysis of variance (ANOVA)], and no difference between genotypes and no interaction between kindling and genotype was detected. Within genotype comparison confirmed a reduction of ADT in kindled TNR−/− mice and revealed a tendency for reduction of ADT in TNR+/+ mice (Fig. 1). No difference between genotypes in postkindling ADTs was found (Fig. 1). The average afterdischarge duration at the beginning of kindling did not differ significantly in the two experimental groups (cf., Fig. 2C).

Figure 1.


ADTs of the amygdala of wild-type (TNR+/+, n = 8) and TNR deficient (TNR−/−, n = 7) mice before (initial ADT) and after (post-ADT) the kindling process. Bars represent mean + SEM. ADTs differ significantly in TNR−/− mice (*, p < 0.05, one-tailed Wilcoxon signed rank test) and tend to decrease in TNR+/+ (+, p = 0.063). ADTs did not differ significantly within each group between prekindling and postkindling.

Figure 2.


Behavioral (A) and electrographic (B and C) seizure parameters during kindling development in wild-type (TNR+/+, n = 8) and TNR deficient (TNR−/−, n = 7) mice. Mice were stimulated once per day. Data in panels A and C are shown as average (±SEM) group data per experimental day (for individual range see text). Kindling development is represented as behavioral seizure stage according to Racine (1972) (A) and afterdischarge duration (C). Examples of electrographic recordings and measurements of afterdischarge duration (between arrows) in TNR+/+ and TNR−/− mice are shown in panel B. The scale bar = 5 s. Two-way ANOVA did not reveal any significant intergroup differences in seizure stage or afterdischarge duration over the period of kindling acquisition, when data were calculated as illustrated in this figure.

Kindling development in TNR+/+ and TNR−/− mice is illustrated in Fig. 2. Both for the progressive increase in seizure stage (Fig. 2A) and afterdischarge duration (Fig. 2C), no significant differences were found between the genotypes by two-way ANOVA, although TNR−/− mice appeared to kindle more slowly than controls. Thus, the kindling process (number of stimulations to first stage 5 seizure) required 7 days (minimum) to 12 days (maximum) in individual TNR+/+ mice as compared with 13 days (minimum) to 18 days (maximum) in individual TNR−/− mice. The expression of seizures was the same in both genotypes, following the description of Racine (1972) (see Materials and Methods).

However, significant differences between TNR−/− and TNR+/+ mice were found when average kindling rates were calculated (Fig. 3). With regard to the number of stimulations needed to reach the kindling criterion (i.e., a fully kindled stage 5 seizure), TNR−/− mice required significantly more stimulations to reach seizure stage 5 than their TNR+/+ littermates (Fig. 3A). Correspondingly, the cumulative seizure duration and cumulative afterdischarge duration to reach the fully kindled state were significantly higher in TNR−/− mice compared with TNR+/+ animals (Fig. 3, B and C), substantiating the difference in the kindling rate. The average duration of seizures and afterdischarges per stimulation did not differ significantly between groups. Average duration of seizures per stimulation was 18.7 ± 2.1 s in TNR+/+ versus 19.6 ± 3.1 s in TNR−/− mice; average afterdischarge duration was 17.0 ± 2.5 s in TNR+/+ versus 18.7 ± 3.1 s in TNR−/− mice.

Figure 3.


Kindling rate shown as number of stimulations (A), cumulative behavioral seizure duration (B), and cumulative afterdischarge duration (C) until fully kindled state in wild-type (TNR+/+, n = 8) and TNR deficient (TNR−/−, n = 7) mice. Bars represent mean + SEM. TNR−/− mice require significantly more stimulations to reach the fully kindled status. This is accompanied by an increased cumulative duration of behavioral and electrographic seizures until the first stage 5 seizure in TNR−/− mice compared with wild-type. ***, p < 0.001, unpaired Student's t-test.

When the stepwise progression of kindling was analyzed by calculating the number of stimulations and the afterdischarge duration that mice spent on average in the different seizure stages during kindling until they reached their first stage 5 seizure, TNR−/− mice stayed significantly longer

in stage 1 and stage 4 than their TNR+/+ littermates (Fig.  4), thus explaining the significant increase in kindling rate illustrated in Fig. 3.

Figure 4.


The stepwise progression of kindling as shown by the number of stimulations (A) and duration of afterdischarges (B) that mice spent on average in the different seizure stages during kindling until they reached their first stage 5 seizure. Data are shown as means ± SEM of eight wild-type (TNR+/+) and seven TNR deficient (TNR−/−) mice, respectively. In contrast to previous figures that provide cumulative data, the parameters shown in this figure were calculated separately for the different seizure stages. TNR−/− mice stayed significantly longer in stage 1 and stage 4 as indicated by increased number of stimulations and increased afterdischarge duration in both stages compared to TNR−/−. Note that especially the duration to progress from stage 4 to stage 5 is retarded in TNR−/− mice, as indicated by the high number of stimulations and long afterdischarge duration that these mice showed in stage 4 before progressing to stage 5. *, p < 0.05 and ****, p < 0.0001, Student's t-test for independent samples.

Kindling-induced morphological changes in TNR-deficient mice

In search of a morphological basis underlying the genotype-related and interindividual variability in kindling susceptibility, we performed morphometric analysis of kindled mice, focusing on two parameters previously found to differ in naive TNR+/+ and TNR−/− mice, such as numbers of astrocytes and PV-positive interneurons (Brenneke et al., 2004a; Morellini, Sivukhina, Stoenica,  Oulianova, Bukalo, Dityatev, Irintchev, Schachner M, unpublished). On the basis of a previous study reporting that mice deficient in S100B kindled more rapidly and exhibited more severe seizures (Dyck et al., 2002), we used S100 as a marker of glial cells.

First, we measured the volume of the DG and found that it was increased in kindled TNR+/+ mice (Fig. 5A), possibly reflecting morphological changes such as edema or astrogliosis. Previously, a 7% increase in volume of the DG was reported for kindled rats (Kotloski et al., 2002). In kindled TNR−/− mice, the volume of the DG was, however, decreased (interaction between kindling and genotype by two-way ANOVA with p = 0.008) (Fig. 5A), suggesting that there is atrophy of this region. There were also significant genotype-independent negative correlations between the volume of the DG and number of stimulations until stage 5 and cumulative afterdischarge duration in stage 1 (ADD1) (Fig. 5, B and C).

Figure 5.


Analysis of the volume of the DG. Bars represent mean values + SEM for the volume of the DG (A) of the TNR+/+ and TNR−/− mice in the different experimental groups (n = 8, 7, 2, 3, 4, 3, respectively). *, p < 0.05, unpaired Student's t-test. Number of stimulations until stage 5 and the cumulative ADD1 correlated negatively with the volume of the DG (B and C, respectively). The Spearman's correlation coefficients (r) and corresponding p values are indicated (n = 7).

Two-way ANOVA revealed no effects of genotype and kindling on the number or density of PV-positive cells when measurements from naive and kindled animals were combined, although there was a higher number of PV-positive cells in naive TNR−/− mice than in TNR+/+ controls (Figs. 6A, 6B,7A, and 7B). Interestingly, we detected a positive correlation between number of PV-positive cells and changes in ADT, measured as a ratio between initial and postkindling ADT in kindled mice independent of genotype (Fig. 7C).

Figure 6.


Immunohistochemical analysis of PV-immunoreactive and S100-immunoreactive cells in the DG. Representative sections showing populations of PV-expressing interneurons in naive TNR+/+ (A) and TNR−/− animals (B), S100-immunoreactive astrocytes in the DG of naive TNR+/+ (C), naive TNR−/− (D), sham TNR+/+ (E), sham TNR−/− (F), kindled TNR+/+ (G), and kindled TNR−/− (H) mice. Notice increased numbers of PV-expressing interneurons in naive TNR−/− mice and increased numbers of S100-expressing astrocytes in sham and kindled animals of both genotypes. Scale bars, 200 μm for major panels and 50 μm for insets, showing stained cells with a higher magnification.

Figure 7.


Analysis of PV-positive (PV+) cells in the DG. Bars represent mean values + SEM for numerical density (A) and total number (B) of PV+ cells in the DG of TNR+/+ and TNR−/− mice in the different experimental groups (n = 8, 7, 2, 3, 4, 3, respectively). *, p < 0.05, unpaired Student's t-test. (C) Ratio of the initial ADT to the post ADT positively correlated with the total number of PV+ interneurons. The Spearman's correlation coefficient (r) and corresponding p value are indicated (n = 7).

Two-way ANOVA revealed a strong effect of kindling on the density and number of S100-expressing (S100+) cells (p < 0.001). There was also a significant effect of genotype on density of S100+ cells (p < 0.05) and a clear tendency for interaction between genotype and kindling (p  =  0.058) (Figs. 6C, 6D, 6E, 6F, 6G, 7A, and 7B). Noteworthy, the numbers of S100+ cells in sham groups increased to the same level in TNR−/− and TNR+/+ mice. An increase in astrocytes after electrode implantation is a known phenomenon (Babb & Kupfer, 2003;  Merriam et  al., 1993; Szarowski et al., 2003). Comparing kindled and sham TNR+/+ groups, it is apparent that kindling led to a reduction in S100+ cells in TNR+/+ mice, as previously reported for rats (Hansen et al., 1990). The higher density of S100+ cells in kindled TNR−/− versus TNR+/+ mice correlated with their higher resistance to kindling (Fig. 8A and 8B). Furthermore, the density of S100+ cells positively correlated with the initial ADT, the cumulative ADD1, and the number of stimulations in stage 4 (Fig. 8C, 8D, and 8E).

Figure 8.


Analysis of S100-expressing (S100+) cells in the DG. Bars represent mean values + SEM for numerical density (A) and the total number (B) of S100+ cells in the DG of TNR+/+ and TNR−/− mice in the different experimental groups (n = 8, 7, 2, 3, 4, 3, respectively). *, p < 0.05 and **, p < 0.01; unpaired Student's t-test. Initial ADT (C), cumulative ADD1 in stage 4 (D), and number of stimulations in stage 4 (E) positively correlated with the density of S100+ cells in the DG. The Spearman's correlation coefficients (r) and corresponding p values are indicated (n = 7).

Discussion

The results of this study show that the stepwise progression of kindling is retarded in TNR−/− mice. Thus, the kindling model used in the present study proved to be more sensitive to detect abnormalities in epileptogenesis in TNR−/− versus TNR+/+ mice than the pilocarpine model (Brenneke et al., 2004a). TNR−/− mice remained significantly longer in stage 1, with mild focal clonus, and stage 4, with bilateral forelimb clonus, than TNR+/+ littermate controls. This indicates that TNR is directly or indirectly involved in the progressive transition of kindled seizure stages at least at two critical time points.

Seizure activity does not spread randomly throughout the brain, but rather is generated and propagated by specific anatomical routes (Löscher & Ebert, 1996a, 1996b; Morimoto et al., 2004). For temporal lobe seizures, there seem to be “preferred” seizure circuits, including the piriform cortex and related sites, which are recruited regardless of the structure initially activated. Burchfiel and Applegate (1989) were the first to propose that kindling is a discontinuous process involving discrete, stepwise transitions from one state of neural organization to another. These transitions act as “gates” controlling the ability of the epileptic discharge to effect the necessary reorganization of neural function that drives the kindling process. At least two critical gates can be defined: a forebrain gate that effects transition from nonconvulsive seizure stages to convulsive stages, and a brainstem gate that effects transition to fully kindled (stage 5) seizures (Löscher & Ebert, 1996a, 1996b). The DG is considered to serve as a gate for passage of electrical activity through the hippocampal-parahippocampal loop and generates paroxysmal activity in response to kindling stimulations. The so-called maximal dentate activation increases the afterdischarge duration and promotes propagation of seizure activity to other limbic and extralimbic areas (Lothman et al., 1991). In this respect, it is noteworthy that we found that the volume of the DG negatively correlated with cumulative afterdischarge duration in stage 1 and number of stimulations until stage 5. Also kindling parameters appeared to be related to numbers of PV- and S100-expressing cells in the DG (see below).

Since kindling can be regarded as a form of neuroplasticity in multiple brain areas including the DG (Maru & Goddard, 1987; Adamec & Young, 2000), it is noteworthy that TNR−/− mice have impaired long-term potentiation (LTP) at CA3-CA1 and perforant path-DG synapses, while LTP at mossy fiber-CA3 and CA3-CA3 synapses is normal (Bukalo et al., 2001; Saghatelyan et al., 2001; Morellini, Sivukhina, Stoenica, Oulianova, Bukalo,  Dityatev, Irintchev, Schachner, unpublished). The deficit in LTP at CA3-CA1 synapses is associated with a deficit in perisomatic inhibition of pyramidal cells in the CA1 region of TNR−/− mice (Saghatelyan et al., 2001; Nikonenko et al., 2003). Furthermore, pharmacological enhancement of GABAergic inhibition restores this form of LTP in the TNR−/− mouse (Bukalo et al., 2007). Since disinhibition of CA1 may lead to episodes of abnormally high excitatory activity in juvenile TNR−/− mice, it is remarkable that seizures evoked by kainate during early postnatal development manifest in reduced capacity for LTP and reduced susceptibility to kindling in adults (Lynch et al., 2000), resembling the phenotype of TNR−/− mice.

In contrast to CA1, impaired synaptic plasticity in the DG of TNR−/− mice is due to elevated  numbers of  perisomatic interneurons and increased perisomatic  GABAergic input to granule cells in this region (Morellini, Sivukhina, Stoenica,  Oulianova, Bukalo,  Dityatev,  Irintchev, Schachner M, unpublished). Because in the present study we did not find a correlation between the number of perisomatic interneurons and progression of kindling, it is most likely that this feature does not contribute to retarded epileptogenesis in TNR−/− mice. Still, it may contribute to kindling-induced changes in the ADT, as suggested by positive correlations between the number of PV-expressing interneurons and the ratio between initial and postkindling ADTs. Alternatively, stronger changes in ADT may increase cell survival and/or expression levels of parvalbumin in interneurons.

Another important feature of TNR−/− mice is an increase in number of S100+ cells as compared to TNR+/+ mice. This genotype-specific difference is not only detected in naive mice but persists in an astrogliotic stage after induction of generalized seizures in mice with implanted electrodes. Thus, not only naive TNR−/− mice show higher levels of S100+ cells than TNR+/+ mice, but also their response to implantation followed by kindling leads to enhanced levels of S100+ cells. The anti-S100 antibody used in the present study is known to label S100B strongly, S100A1 weakly, S100A6 very weakly, and does not label S100A2, S100A3, or S100A4 (Ilg et al., 1996). Thus, it detects in the brain predominantly S100B protein, a 20 kDa Ca2+ and Zn2+ binding homodimer. In the mouse, S100 immunostaining with the antibody used in the present study is confined to astrocytes, whereas neurons are not labeled (Irintchev et al., 2005). An increase in number of S100+ cells in naive TNR−/− mice is in agreement with previous studies reporting elevated numbers of GFAP-positive astrocytes in naive TNR−/− mice (Brenneke et al., 2004a). Furthermore, our analysis revealed that progression of epileptogenesis negatively correlated with the number of cells expressing S100. As any correlation, this finding does not imply a causative relationship between two phenomena. However, our finding showing a slower progression of epileptogenesis in TNR−/− mice (having higher numbers of S100+ cells) complements a previous study reporting a more rapid onset and more severe seizures in kindled mice deficient in S100B as compared with wild-type mice (Dyck et al., 2002). Thus, S100B is likely one of the astrocyte-related traits contributing to retarded epileptogenesis in TNR−/− mice. S100B may exert its action by buffering of Ca2+ and Zn2+ in astrocytes or by being secreted and exerting a trophic effect and/or stimulate Ca2+ uptake in neurons and astrocytes (Winningham-Major et al., 1989; Barger & van Eldik, 1992). Contributions of other astrocytic factors to the reduced rate of slower epileptogenesis in TNR−/− mice are also  conceivable.

Interestingly, there are other examples of extracellular matrix components, related to TNR, which affect epileptogenesis. Enzymatic removal of hyaluronan by treatment with hyaluronidase reduced kainate-induced hippocampal mossy fiber sprouting, one of the hallmarks associated with temporal lobe epilepsy (Bausch, 2006). Another hallmark of temporal lobe epilepsy is granule cell dispersion (i.e., a widening of the granule cell layer in the DG). In the kainate-injected hippocampus, development of granule cell dispersion is associated with a decrease in messenger RNA (mRNA) synthesis of the extracellularly secreted protein reelin, and neutralization of reelin by application of the reelin-specific CR-50 antibody mimics this phenotype (Heinrich et al., 2006). These data, our present work, and several studies highlighting the seizure-induced changes in expression of TNR and other extracellular matrix molecules (Nakic et al., 1996; Represa & Ben-Ari, 1997; Brenneke et al., 2004b; Heck et al., 2004) support the idea that remodeling of the extracellular matrix is involved in different aspects of epileptogenesis and encourage approaches to therapeutically target extracellular matrix molecules to restrain harmful structural changes and/or promote regeneration during interseizure intervals in epileptic patients.

Acknowledgments

K.H. and E.S. contributed equally to this work. This work was supported by the Deutsche Forschungsgemeinschaft (Di 702/4-1,2,3 to A.D. and SPP 1172 to M.S.). M.S. is New Jersey Professor of Spinal Cord Research. We thank Andrey Irintchev for helpful suggestions, Emanuela Szpotowicz for technical assistance, and Fabio Morellini and Achim Dahlmann for supervising of breeding and genotyping of mice.

Conflict of interest: We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors have no conflicts of interest to disclose.

Ancillary