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Suppressed Kindling Epileptogenesis and Perturbed BDNF and TrkB Gene Regulation in NT-3 Mutant Mice
EXPERIMENTAL NEUROLOGY ARTICLE NO.
145, 93–103 (1997)
EN976478
Suppressed Kindling Epileptogenesis and Perturbed BDNF and TrkB Gene Regulation in NT-3 Mutant Mice Eskil Elme´r, Merab Kokaia, Patrik Ernfors,* Istvan Ferencz, Zaal Kokaia, and Olle Lindvall Department of Clinical Neuroscience, Section of Restorative Neurology, Wallenberg Neuroscience Center, University Hospital, S-221 85 Lund, Sweden; and *Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden
activates mainly the TrkC receptor, NGF is the specific ligand for TrkA, and BDNF and NT-4/5 interact with the TrkB receptor. NT-3 can probably function both locally (40) and as a classical target-derived trophic factor in the central nervous system (CNS) (8). Studies in vitro have shown that NT-3 promotes the survival and morphological differentiation of specific populations of embryonic neurons from the hippocampus (20), striatum (62), substantia nigra (19, 57), locus coeruleus (15), and cerebellum (44). Furthermore, NT-3 has been reported to protect cultured hippocampal, cortical, and striatal neurons against glucose deprivation-induced cell death (7, 45). However, recent data indicate that although NT-3 attenuates apoptotic death, this neurotrophin can potentiate necrotic death of cortical neurons in vitro (26). Administration of NT-3 to adult rats prevents the death of locus coeruleus neurons after neurotoxic damage (1) and stimulates sprouting of the corticospinal tract (52). In addition, intraventricular infusion of NT-3 improves memory deficits in aged rats (14). To what extent these findings reveal the physiological action of endogenous NT-3 in the CNS is unclear. Mutant mice lacking the NT-3 or TrkC gene show severe impairment of proprioception and neuronal loss in sympathetic and sensory ganglia but no obvious morphological abnormalities in the brain (11, 13, 59, 60). Various stimuli influence NT-3 gene expression in mature central neurons. Stress induces increased NT-3 mRNA levels in the dentate gyrus, hippocampus, and locus coeruleus (55, 56). Brain insults such as epileptic seizures (3, 50, 51), cerebral ischemia, and hypoglycemic coma (29, 32) downregulate NT-3 mRNA expression in dentate granule cells. In addition, stimulations inducing long-term potentiation increase NT-3 mRNA levels in CA1 pyramidal neurons (47) and reduce expression in dentate granule cells (6). The rapid changes of gene expression for NT-3 as well as for NGF and BDNF in response to seizure activity have suggested a role for these factors in epileptogenesis (33), which is supported by data obtained in the
In the kindling model of epilepsy, repeated electrical stimulations lead to progressive and permanent intensification of seizure activity. We find that the development of amygdala kindling is markedly retarded in mice heterozygous for a deletion of the neurotrophin-3 (NT-3) gene (NT-31/2 mice). These mice did not reach the fully kindled state (3rd grade 5 seizure) until after 28 6 4 days of stimulation compared to 17 6 2 days in the wild-type animals. The deficit in the NT-31/2 mice reflected dampening of the progression from focal to generalized seizures. The number of stimulations required to evoke focal (grade 1 and 2) seizures did not differ between the groups, but the NT-3 mutants spent a considerably longer period of time (13 6 3 days) than wild-type mice (2 6 1 days) in grade 2 seizures. As assessed by test stimulation 4–12 weeks after the 10th grade 5 seizure, kindling was maintained in the NT-3 mutants. In situ hybridization showed 30% reduction of basal NT-3 mRNA levels and lack of upregulation of TrkC mRNA expression at 2 h after a generalized seizure in dentate granule cells of the NT-31/2 mice, whereas the seizure-evoked increase in brain-derived neurotrophic factor (BDNF) and TrkB mRNA levels was enhanced. These results indicate that endogenous NT-3 levels can influence the rate of epileptogenesis, and suggest a link between NT-3 and BDNF gene regulation in dentate granule cells. r 1997 Academic Press
INTRODUCTION
Neurotrophin-3 (NT-3) is a member of the neurotrophin family of trophic molecules, which also comprises nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and neurotrophin-6 (18, 31). In the adult rat brain, high expression of NT-3 mRNA is only detected in dentate granule cells and in the CA2 and medial CA1 pyramidal layer (12, 36). The highest levels of NT-3 protein are found in the hippocampus and piriform cortex (58). The biological activities of the neurotrophins are mediated through the Trk tyrosine kinase receptors (2). NT-3 93
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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kindling model. In kindling, repeated electrical stimulation of discrete brain areas leads to progressive and permanent intensification of seizure activity (38). During the development of kindling, each stimulus-evoked seizure causes increased NGF and BDNF synthesis in cortical and hippocampal neurons and a reduction of NT-3 mRNA expression in dentate granule cells (3, 4, 9). Recent findings indicate that endogenously produced NGF and BDNF promote kindling epileptogenesis. Heterozygous BDNF knockout mice show markedly delayed kindling development (27), which is also observed after intraventricular administration of an antibody to NGF (16, 61). In the present study, we have analyzed kindling development and neurotrophin and Trk gene expression in mice carrying a deletion of the NT-3 gene. Because homozygous NT-3 mutant mice (NT-32/2) die soon after birth, only heterozygotes (NT-31/2) were used. The objectives were twofold: first, to determine whether NT-3 is involved in epileptogenesis; second, to explore the possibility that the presumed deficit in the NT-3 system in the mutant mice influences gene regulation of another neurotrophin. MATERIALS AND METHODS
Animals and kindling procedure. Heterozygous NT-3 mutant mice were generated as described previously (11). The animals were housed under 12-h light/ 12-h dark conditions with ad libitum access to water and food. Adult male NT-31/1 (n 5 14) and NT-31/2 (n 5 13) mice, weighing 28–32 g at the start of the experiment, were used. All animals were anesthetized with Equithesin (3.0 ml/kg body wt ip) and bipolar stainless steel electrodes were implanted bilaterally into the amygdala (coordinates: toothbar at 23.3; 1.5 mm caudal to bregma; 3.0 mm lateral to midline; and 4.0 mm ventral to dura). Two weeks later, kindling stimulations (1 ms pulses, 100 Hz frequency, 1 s duration) once daily in the left amygdala were started in 10 NT-31/1 and 8 NT-31/2 mice and were continued until the animals had experienced 10 grade 5 generalized seizures. Four NT-31/1 and 5 NT-31/2 mice were nonkindled. On the first day of stimulation, the current intensity was gradually increased from 20 µA stepwise by 10 µA to determine the threshold for eliciting focal epileptiform activity (afterdischarge) of more than 5 s duration in the amygdala. This current intensity was used in subsequent stimulations. The electroencephalogram was recorded from both electrodes, and behavioral convulsions were scored blindly according to a modification of the scale of Racine (48): grade 1, facial twitches; grade 2, chewing and nodding; grade 3, forelimb clonus; grade
4, rearing, body jerks, tail upholding; and grade 5, imbalance, hind limb clonus, vocalization. Four to 12 weeks after the 10th grade 5 seizure, all stimulated mice were tested for the permanence of the kindling phenomenon. The threshold for seizure induction was determined by gradually raising the stimulation intensity similar to the initial kindling protocol, and when an afterdischarge had been induced, seizure grade and other related parameters were recorded. Two hours after the test stimulation-induced seizure, animals were anesthetized and decapitated and the brains were immediately frozen on dry ice. As assessed in cresyl violet-stained sections, the stimulating electrode was in all animals localized in the amygdaloid complex without any difference between the groups. In situ hybridization and image analysis. Unless otherwise stated, all chemicals were obtained from Sigma (Sigma-Aldrich Sweden). Cryostat sections (14 µm) were taken through the dorsal hippocampus (at a level 1.5–1.9 mm caudal to bregma) of NT-31/1 and NT-31/2 mice, which were either nonkindled and electrode-implanted (n 5 4 and n 5 5 in each group, respectively) or kindled (n 5 5, randomly selected in each group). The sections were then processed for in situ hybridization as described previously (3, 12). NT-3, BDNF, and TrkC mRNAs were detected using oligonucleotide probes complementary to nucleotides 667–717, 746–795 (12), and 1186–1236 (40) of the corresponding rat cDNA sequences, respectively. The oligonucleotide probe for full-length TrkB was complementary to nucleotides 1905–1952 of the mouse TrkB cDNA sequence (25). The probes were labeled at their 38-ends with [a-35S]dATP using terminal deoxyribonucleotidyl transferase (Amersham) to a specific activity of approximately 109 cpm/µg. After labeling, all probes were purified using Nensorb 20 columns (Dupont Medical Scandinavia). Ten millimolar dithiothreitol (DTT) was added to the labeled probes, which were then stored at 220°C. After fixation, the sections were rinsed three times during 5 min with phosphate-buffered saline (PBS), dehydrated, and air-dried. Hybridization was performed in 50% formamide, 43 SSC, 13 Denhardt’s solution, 10% dextran sulfate, 0.5 mg/ml sheared and denatured salmon sperm DNA, 1% sarkosyl (N-lauroyl sarcosine), 0.02 M phosphate buffer (PB; pH 7.0), and 0.1 M DTT using 107 cpm/ml of the respective probe. The sections were hybridized overnight at 42°C in a humidified chamber with parafilm coverslips and subsequently washed four times (15 min each) at 55°C in 13 SSC. After washing, all sections were dehydrated, air-dried, and, together with radioactive isotope standards (14C micro-scales; Amersham), exposed onto b-max X-ray film (Amersham) for 10–14 days. For cellular resolution, slides were dipped in Ilford K5 liquid photo-
KINDLING IN NT-3 MUTANT MICE
emulsion (diluted 1:1 in water), exposed for 4–6 weeks, developed in Kodak D-19, and lightly counterstained with hematoxylin–eosin. Quantification of hybridization signals was performed blindly by computerized image analysis using Image 1.57 software (Wayne Rasband, NIMH). Gray levels from the standards were used in a third degree polynomial calibration to obtain equivalent values of tissue radioactivity (nCi/g) from optical densities measured on the films. Background optical densities were subtracted from the hybridization signals. In every section, two to three measurements were made in the brain region of interest. Three sections were analyzed for each brain, and the mean value of the measurements was used for the statistical analysis. Morphological analysis. Cresyl violet-stained coronal sections (14 µm; alternate sections to those for in situ hybridization) were used to study gross morphological characteristics in the forebrain of NT-31/1 and NT-31/2 nonkindled (n 5 4 and n 5 5, respectively) and kindled (n 5 5 in each group) mice. All light microscopical analyses were performed blindly. The thickness of the dentate gyrus, CA1 and CA3 regions, and piriform and parietal cortices was measured using 103 or 403 objective. We also counted the total number of neurons in the dentate gyrus hilus and the cell numbers in 0.036-mm2 areas in the dentate granule cell layer, CA1 and CA3 pyramidal layers, basolateral amygdaloid nucleus, and piriform and parietal cortices. The analysis was carried out at comparable levels and areas in the different animals. For each region and animal, one value was obtained which represents the mean of three measurements or cell counts per section in three sections. This value was considered as one data entry in the statistical comparison between groups. Statistical evaluation. Kindling parameters in the different groups were analyzed using Student’s unpaired t test and autoradiographic and morphological data using one-way ANOVA followed by Bonferroni’s post hoc test. Student’s paired t test was performed to compare thresholds for seizure induction during kindling and test stimulations, and Kendall rank correlation was used for correlation analyses. The data are presented as means 6 SEM, and differences are considered significant at P , 0.05. RESULTS
Kindling Development Is Delayed but Maintenance Is Normal in NT-3 Mutants The behavioral expression of the different seizure grades was the same in the two strains and closely resembled that previously described in rats (48). In contrast, the development of amygdala kindling was significantly retarded in the heterozygous NT-3 mutant
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FIG. 1. Delayed kindling development in NT-31/2 mice. Bars represent mean number of amygdala stimulations to reach the different seizure grades during kindling in NT-31/1 (n 5 10) and NT-31/2 mice (n 5 8). (*) Significantly different from NT-31/1, P , 0.05; Student’s unpaired t test.
mice (Fig. 1). These mice did not exhibit the first grade 5 seizure until after 26 6 4 days of stimulation compared to 15 6 2 days in the wild-type mice (P , 0.05, Student’s unpaired t test). The fully kindled state (third grade 5 seizure) was reached after 28 6 4 days in the NT-31/2 and 17 6 2 days in the NT-31/1 mice. The deficit in the NT-3 mutants mainly reflected dampening of the progression from focal to generalized seizures. There was no significant difference between the groups in the number of stimulations required to evoke the first grade 1 or 2 focal seizures (Fig. 1). In contrast, the NT-3 mutants spent a considerably longer period of time (13 6 3 days) than wild-type mice (2 6 1 days) in grade 2 seizures. Seizure threshold, duration of afterdischarge at various stages, and latency to and duration of behavioral convulsions did not differ significantly between the groups (Table 1). In order to investigate whether the NT-3 gene mutation had influenced the permanence of the kindling phenomenon, NT-31/1 and NT-31/2 mice were left unstimulated for 4–12 weeks after having experienced 10 grade 5 seizures. When a test stimulus was subsequently given, the animals in both groups responded with a generalized seizure, indicating that the kindled state was maintained. There was no difference in seizure grade between wild-type mice and NT-3 mutants (4 6 0.9 and 4 6 0.5, respectively). The threshold to induce seizure, afterdischarge duration, and latency to and duration of behavioral convulsions were also similar in the two groups (Table 2). Furthermore, there
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TABLE 1 Seizure Characteristics during Kindling Development in NT-3 Mutant and Wild-type Mice
Groups
Seizure threshold (µA)
Afterdischarge duration at different grades (s) 1
2
3
4
Behavioral convulsions 5
Latency (s)
Duration (s)
NT-31/1
50 6 5
20 6 2
21 6 2
26 6 2
32 6 4
30 6 3
12 6 1
18 6 1
NT-31/2
45 6 6
25 6 5
26 6 4
28 6 3
33 6 4
38 6 5
14 6 1
18 6 3
Note. Latency to and duration of behavioral convulsions were measured for grade 4–5 seizures. Values represent mean 6 SEM. No significant differences were found between NT-3 mutants and wild-type mice; P . 0.05, Student’s unpaired t test (n 5 10 and n 5 8 in the NT-31/1 and NT-31/2 groups, respectively).
was no significant difference in the threshold to induce seizures during kindling as compared to that during test stimulation in either the NT-3 mutants or the wild-type animals. Basal Expression of NT-3 mRNA Is Reduced and Regulation of TrkC mRNA after Seizures Is Perturbed in NT-3 Mutants The distribution of mRNA for NT-3 in nonkindled animals, as assessed using in situ hybridization, agreed well with previous findings in the rat (12, 36), and did not differ between the mutants and the wild-type mice. Abundant NT-3 mRNA expression was only observed in the dentate granule cell layer and in the CA2 and medial CA1 pyramidal layers, suggesting that the spatial control of NT-3 mRNA expression is not perturbed in the NT-31/2 mice. We quantified NT-3 mRNA levels in the dentate granule cell layer and in the medial CA1 and CA2 pyramidal layers using computerized image analysis. In control animals, not subjected to the kindling procedure, basal expression of NT-3 mRNA in dentate granule cells was reduced by 30% in NT-31/2 compared to that in NT-31/1 mice (P , 0.05; one-way ANOVA followed by Bonferroni’s TABLE 2 Seizure Characteristics during Test Stimulation 4–12 Weeks after Kindling in NT-3 Mutant and Wild-type Mice Behavioral convulsions
Groups
Mean seizure grade
Afterdischarge duration (s)
Seizure threshold (µA)
Latency (s)
NT-31/1
4 6 0.9
31 6 1
42 6 9
22 6 2
963
NT-31/2
4 6 0.5
54 6 9
49 6 7
29 6 6
29 6 6
Duration (s)
Note. Values represent mean 6 SEM. No significant differences were found between NT-3 mutants and wild-type mice; P . 0.05, Student’s unpaired t test (n 5 5 and n 5 8 in NT-31/1 and NT-31/2 groups, respectively; five mice in the control group were excluded due to loss of the electrodes).
post hoc test; Figs. 2A and 3). In the medial CA1 and CA2 pyramidal layers also, basal NT-3 mRNA levels were slightly (18%) lower in the NT-3 mutants (not statistically significant). At 2 h after the last generalized seizure, NT-3 mRNA levels in the wild-type mice had decreased significantly (by 75%) in dentate granule cells. A similar reduction (by 73%) occurred in NT-31/2 mice. Although the mean seizure-induced NT-3 mRNA expression in NT-31/2 mice was 28% lower compared to that in wild-type animals, this difference was not statistically significant (P . 0.05; Figs. 2A and 3). No change in NT-3 mRNA levels was detected after seizures in the CA2 and medial CA1 pyramidal layers in either group (data not shown). In order to explore the possibility that the presumed low levels of NT-3 in the mutants might have influenced the expression of the TrkC receptor, we also measured TrkC mRNA levels in the dentate granule cell layer, CA1, CA2 and CA3 pyramidal layers, parietal cortex, piriform cortex, and basolateral amygdaloid nucleus. The nonkindled NT-31/2 mice did not differ from wild-type animals in basal TrkC mRNA expression (Fig. 5B). Similar to previous observations in the rat (3), the wild-type mice exhibited a marked increase in TrkC mRNA levels (to 173% of nonkindled control) at 2 h after the last generalized seizure. In contrast, no increase in TrkC mRNA expression was detected at this timepoint in the dentate granule cell layer of the NT-3 mutants (Figs. 5B and 6B). No changes of TrkC mRNA expression were found after seizures outside the dentate gyrus (data not shown). There was no significant correlation between the time from the 10th grade 5 seizure to the test stimulation (4–12 weeks) and the seizure-evoked NT-3 or TrkC mRNA expression in the different animals. Seizure-Induced Expression of BDNF and TrkB mRNAs Is Enhanced in NT-3 Mutants The presumed deficit in the NT-3 system in the mutants could hypothetically affect the expression of other neurotrophins or their receptors. We therefore analyzed levels of mRNA for BDNF and its high-affinity
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increased expression of BDNF mRNA after generalized seizures in all regions analyzed (ranging between 131 and 350% of control; see Figs. 2B and 4). However, the seizure-induced level of BDNF mRNA in dentate granule cells in the mutant mice was 130% higher compared to the level in kindled, wild-type animals (P , 0.05, one-way ANOVA followed by Bonferroni’s post hoc test; Figs. 2B and 4). No difference between NT-31/1 and NT-31/2 mice in BDNF mRNA expression after seizures was observed in any other brain area (data not shown). The NT-3 mutant mice did not differ from wild-type animals in basal TrkB mRNA levels (Figs. 5A and 6A). Two hours after the last generalized seizure, a significant increase in TrkB mRNA expression (to 471% of control) was observed in the dentate gyrus of NT-31/1 mice (Figs. 5A and 6A). Similar to BDNF mRNA expression, the seizure-induced TrkB mRNA levels in this region were significantly higher (38%) in NT-31/2 compared to NT-31/1 mice. There were no differences between NT-3 mutants and wild-type mice in seizureevoked TrkB mRNA levels outside the dentate gyrus (data not shown). Since there was a trend for the afterdischarge during the last seizure to be longer in the NT-3 mutants (not statistically significant, P . 0.05), we explored whether the higher expression of BDNF and TrkB mRNAs in these animals might be dependent on the duration of seizure activity. However, there was no statistically significant correlation between either the BDNF or the TrkB mRNA levels and the afterdischarge duration in the individual NT-31/1 and NT-31/2 mice. Furthermore, the seizure-evoked BDNF and TrkB mRNA levels in the different animals did not correlate with the time interval between kindling and test stimulation. Gross Anatomy Is Normal in NT-3 Mutants
FIG. 2. Reduced basal NT-3 and increased seizure-induced BDNF mRNA levels in the dentate granule cell layer of NT-31/2 mice. Levels of (A) NT-3 and (B) BDNF mRNA under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) as quantified using image analysis on in situ hybridization autoradiograms. (*) Significant difference, P , 0.05, one-way ANOVA followed by Bonferroni’s post-hoc test.
receptor, TrkB, in the dentate granule cell layer, hippocampal CA1 and CA3 regions, amygdala, and piriform and parietal cortices of nonkindled controls and in animals 2 h after the last generalized seizure. Basal expression of BDNF mRNA in NT-31/2 mice was not different from that in NT-31/1 mice in any of these regions (see Figs. 2B and 4). Both groups showed
Examination of cresyl violet-stained sections revealed no significant differences between NT-31/1 and NT-31/2 mice in the layering or thickness of the dentate gyrus, CA1 and CA3 regions, and piriform and parietal cortices. Furthermore, the total cell number in the dentate gyrus hilus and the cell numbers in 0.036mm2 areas in the dentate granule cell layer, CA1 and CA3 pyramidal layers, basolateral amygdaloid nucleus, and piriform and parietal cortices did not differ between the mutant and the wild-type mice either in nonkindled or in kindled animals (data not shown). DISCUSSION
This study shows that the rate of amygdala kindling is significantly retarded, while the persistence of the kindled state is not affected, in mice heterozygous for a deletion of the NT-3 gene. Furthermore, in these ani-
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FIG. 3. Examples of in situ hybridization autoradiograms demonstrating the levels of NT-3 mRNA expression under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in the hippocampus of NT-31/2 and NT-31/1 mice.
mals the seizure-evoked increases in BDNF and TrkB mRNA expression in dentate granule cells are enhanced compared to those in wild-type mice. The present findings suggest that endogenous NT-3 levels can influence seizure development during epileptogenesis and that a deficit in the function of this neurotrophin may lead to changes in gene regulation of another neurotrophin, BDNF, and its high-affinity receptor, TrkB. Previous in situ hybridization studies (9, 21, 41) and measurements of NGF (4), BDNF (46), and TrkB protein (41) content indicate that basal and seizureinduced expression of neurotrophin and Trk mRNAs to a large extent reflects the level of the corresponding proteins. We observed that under basal conditions, NT-3 mRNA expression was reduced by 30% in the dentate granule cell layer of NT-31/2 compared to NT-31/1 mice. Also at 2 h after a generalized seizure, NT-3 mRNA levels seemed to be lower in the mutants, but this difference was not statistically significant. The reduction of NT-3 mRNA expression after brain insults had not reached its maximum at 2 h (32), and the difference between NT-3 mutants and wild-type animals may, therefore, be more pronounced at later timepoints. In any case, since the seizure-evoked increase in TrkC mRNA expression detected in NT-31/1 mice was not observed in the mutants, the present results suggest that the NT-3 system in the dentate granule cell layer of NT-31/2 mice is impaired both under basal conditions and after seizures. We could not detect any significant hybridization signal outside the hippocampus in the two strains, which is in agreement with previous findings in the
adult rat (12). However, RNA blot analysis has revealed a weak signal for NT-3 in cerebral cortical tissue (10), and with ELISA a relatively high level of NT-3 protein has been detected in the piriform cortex and low levels in other cortical areas (58). These data suggest that NT-3 may influence the function of the cerebral cortex also in the adult, in particular, since the TrkC receptor is abundantly expressed in this region (40). Given the effects of NT-3 on excitatory synaptic transmission in cultured cortical neurons (24), it cannot be excluded that a reduction of basal and seizure-induced levels of NT-3 protein in the cerebral cortex of the NT-31/2 mice might have contributed to the observed deficit in kindling development. It seems highly warranted to explore this possibility by measurement of NT-3 protein levels in the different CNS regions of the mutants. The effect of NT-3 on seizure development during kindling epileptogenesis might be due to a direct action of this neurotrophin on synaptic transmission. It has been shown that NT-3 enhances acetylcholine release at the Xenopus neuromuscular junction (34) and that direct injection of NT-3 into the hippocampus gives rise to epileptiform and theta-like activities, which can be blocked by the muscarinic receptor antagonist, scopolamine (5). Glutamatergic transmission at the Schaffer collateral-CA1 synapses is potentiated for 2 to 3 h after application of NT-3 to hippocampal slices (23). Furthermore, NT-3 specifically enhances impulse activity of cortical neurons in culture and induces synchronization of excitatory synaptic currents (24), probably through reduction of GABAergic synaptic transmission. The presumed reduced NT-3 protein levels in the mutant mice might lead to less inhibition of GABAergic
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FIG. 4. Examples of in situ hybridization autoradiograms (A) and bright-field photomicrographs (B) depicting BDNF mRNA levels in the dentate granule cell layer under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in NT-31/2 and NT-31/1 mice. Bar, 20 µm.
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FIG. 5. Perturbed seizure-induced TrkB and TrkC mRNA expression in the dentate granule cell layer of NT-31/2 mice. Levels of (A) TrkB and (B) TrkC mRNA under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in NT-31/2 and NT-31/1 mice as quantified using image analysis on in situ hybridization autoradiograms. (*) Significant difference, P , 0.05, one-way ANOVA followed by Bonferroni’s post-hoc test.
transmission and a subsequent reduction of neuronal activity and synchronization, resulting in delayed epileptogenesis. In support of this hypothesis, synchronization of neuronal activity appears to be crucial for generating epileptic events (35, 42), and reduction of excitatory glutamatergic (39, 43) or increase in inhibi-
tory GABAergic transmission (22, 53, 54, 64) leads to suppressed seizure development. Another possibility is that the retardation of kindling rate in the mutants is due to reduced levels of NT-3 during CNS development, causing abnormalities in the formation of anatomical connections or brain regions important for seizure generalization. NT-3 has been shown to promote neuronal differentiation of cortical and hippocampal precursor cells (17, 63) and to support survival and differentiation of noradrenergic locus coeruleus neurons (1, 15). Arguing against a morphological deficit in the NT-31/2 mutants, the spread of seizure activity from the stimulated to the nonstimulated amygdala and the expression of behavioral convulsions were unchanged in these animals compared to wildtype mice. Furthermore, a dysfunction of the noradrenergic system should, most likely, have facilitated and not delayed kindling epileptogenesis (37). Finally, we did not detect any morphological abnormalities in the NT-31/2 mice, which is in agreement with previous observations in the forebrain of NT-32/2 mice (11, 25). The enhancement of the seizure-evoked increase in BDNF and TrkB mRNA expression in NT-31/2 mice was confined to dentate granule cells. BDNF and NT-3 probably act on the same dentate granule cells, most of which express both the TrkB and the TrkC receptor (28). It may be hypothesized that BDNF and NT-3 mechanisms in these cells are linked and that a deficit in the activation of the TrkC receptor causes an upregulation of BDNF and TrkB synthesis. Supporting the idea that one neurotrophin may influence the regulation of another neurotrophin, BDNF and NT-4 (which both act on the TrkB receptor) increase NT-3 mRNA expression in hippocampal neurons (30). Hypothetically, the balance between the BDNF and the NT-3 systems after seizures, suggested by our data, may be important for the fine tuning of synaptic efficacy in the hippocampus. However, the present findings suggest that the presumed increase in the function of the BDNF system after seizures is insufficient to preserve normal excitability and kindling development in NT-3 mutants. In our previous study (27), heterozygous BDNF knockout mice, which had significantly reduced BDNF mRNA levels in dentate granule cells but exhibited no perturbation of NT-3 mRNA regulation after seizures, showed marked inhibition of kindling epileptogenesis. The less pronounced retardation of kindling development in the NT-31/2 compared to the BDNF1/2 mice could, at least partly, be attributable to the enhanced seizure-induced BDNF and TrkB synthesis in the dentate granule cells of the NT-3 mutants. In conclusion, the present study provides evidence that NT-3, similar to what has recently been reported for NGF (16, 61) and BDNF (27), can influence kindling epileptogenesis. For NGF, this effect may be exerted through the induction of mossy fiber sprouting (49, 61),
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FIG. 6. Examples of in situ hybridization autoradiograms demonstrating TrkB (A) and TrkC (B) mRNA expression under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in the hippocampus of NT-31/2 and NT-31/1 mice.
whereas it seems more likely that NT-3 and BDNF can act via a direct effect on synaptic transmission. Kindling is an animal model of the most common type of epilepsy in adult humans, complex partial seizures (38), and kindling-like processes probably contribute to epileptogenesis in patients. Our data suggest that the downregulation of NT-3 synthesis after seizures and other brain insults, such as traumatic injury, can act to dampen the development of epilepsy. Furthermore, they suggest that strategies
leading to the attenuation of neurotrophin levels after these insults might counteract epileptogenesis in humans. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council, the Swedish Society for Medical Research, the Medical Faculty, University of Lund, the Royal Physiographic Society, and the Wiberg, Kock, Elsa Schmitz, and Elsa & Thorsten
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Segerfalk foundations. In addition, P.E. was supported by the Swedish Cancer Society and the Augusta and Petrus Hedlunds foundation. M.K. and Z.K. were partly supported by funds from the I. Beritashvili Institute of Physiology, Georgian Academy of Sciences. We are grateful to Marie Lundin for secretarial work and to Agneta Othberg for skillful technical assistance.
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145, 93–103 (1997)
EN976478
Suppressed Kindling Epileptogenesis and Perturbed BDNF and TrkB Gene Regulation in NT-3 Mutant Mice Eskil Elme´r, Merab Kokaia, Patrik Ernfors,* Istvan Ferencz, Zaal Kokaia, and Olle Lindvall Department of Clinical Neuroscience, Section of Restorative Neurology, Wallenberg Neuroscience Center, University Hospital, S-221 85 Lund, Sweden; and *Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden
activates mainly the TrkC receptor, NGF is the specific ligand for TrkA, and BDNF and NT-4/5 interact with the TrkB receptor. NT-3 can probably function both locally (40) and as a classical target-derived trophic factor in the central nervous system (CNS) (8). Studies in vitro have shown that NT-3 promotes the survival and morphological differentiation of specific populations of embryonic neurons from the hippocampus (20), striatum (62), substantia nigra (19, 57), locus coeruleus (15), and cerebellum (44). Furthermore, NT-3 has been reported to protect cultured hippocampal, cortical, and striatal neurons against glucose deprivation-induced cell death (7, 45). However, recent data indicate that although NT-3 attenuates apoptotic death, this neurotrophin can potentiate necrotic death of cortical neurons in vitro (26). Administration of NT-3 to adult rats prevents the death of locus coeruleus neurons after neurotoxic damage (1) and stimulates sprouting of the corticospinal tract (52). In addition, intraventricular infusion of NT-3 improves memory deficits in aged rats (14). To what extent these findings reveal the physiological action of endogenous NT-3 in the CNS is unclear. Mutant mice lacking the NT-3 or TrkC gene show severe impairment of proprioception and neuronal loss in sympathetic and sensory ganglia but no obvious morphological abnormalities in the brain (11, 13, 59, 60). Various stimuli influence NT-3 gene expression in mature central neurons. Stress induces increased NT-3 mRNA levels in the dentate gyrus, hippocampus, and locus coeruleus (55, 56). Brain insults such as epileptic seizures (3, 50, 51), cerebral ischemia, and hypoglycemic coma (29, 32) downregulate NT-3 mRNA expression in dentate granule cells. In addition, stimulations inducing long-term potentiation increase NT-3 mRNA levels in CA1 pyramidal neurons (47) and reduce expression in dentate granule cells (6). The rapid changes of gene expression for NT-3 as well as for NGF and BDNF in response to seizure activity have suggested a role for these factors in epileptogenesis (33), which is supported by data obtained in the
In the kindling model of epilepsy, repeated electrical stimulations lead to progressive and permanent intensification of seizure activity. We find that the development of amygdala kindling is markedly retarded in mice heterozygous for a deletion of the neurotrophin-3 (NT-3) gene (NT-31/2 mice). These mice did not reach the fully kindled state (3rd grade 5 seizure) until after 28 6 4 days of stimulation compared to 17 6 2 days in the wild-type animals. The deficit in the NT-31/2 mice reflected dampening of the progression from focal to generalized seizures. The number of stimulations required to evoke focal (grade 1 and 2) seizures did not differ between the groups, but the NT-3 mutants spent a considerably longer period of time (13 6 3 days) than wild-type mice (2 6 1 days) in grade 2 seizures. As assessed by test stimulation 4–12 weeks after the 10th grade 5 seizure, kindling was maintained in the NT-3 mutants. In situ hybridization showed 30% reduction of basal NT-3 mRNA levels and lack of upregulation of TrkC mRNA expression at 2 h after a generalized seizure in dentate granule cells of the NT-31/2 mice, whereas the seizure-evoked increase in brain-derived neurotrophic factor (BDNF) and TrkB mRNA levels was enhanced. These results indicate that endogenous NT-3 levels can influence the rate of epileptogenesis, and suggest a link between NT-3 and BDNF gene regulation in dentate granule cells. r 1997 Academic Press
INTRODUCTION
Neurotrophin-3 (NT-3) is a member of the neurotrophin family of trophic molecules, which also comprises nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and neurotrophin-6 (18, 31). In the adult rat brain, high expression of NT-3 mRNA is only detected in dentate granule cells and in the CA2 and medial CA1 pyramidal layer (12, 36). The highest levels of NT-3 protein are found in the hippocampus and piriform cortex (58). The biological activities of the neurotrophins are mediated through the Trk tyrosine kinase receptors (2). NT-3 93
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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kindling model. In kindling, repeated electrical stimulation of discrete brain areas leads to progressive and permanent intensification of seizure activity (38). During the development of kindling, each stimulus-evoked seizure causes increased NGF and BDNF synthesis in cortical and hippocampal neurons and a reduction of NT-3 mRNA expression in dentate granule cells (3, 4, 9). Recent findings indicate that endogenously produced NGF and BDNF promote kindling epileptogenesis. Heterozygous BDNF knockout mice show markedly delayed kindling development (27), which is also observed after intraventricular administration of an antibody to NGF (16, 61). In the present study, we have analyzed kindling development and neurotrophin and Trk gene expression in mice carrying a deletion of the NT-3 gene. Because homozygous NT-3 mutant mice (NT-32/2) die soon after birth, only heterozygotes (NT-31/2) were used. The objectives were twofold: first, to determine whether NT-3 is involved in epileptogenesis; second, to explore the possibility that the presumed deficit in the NT-3 system in the mutant mice influences gene regulation of another neurotrophin. MATERIALS AND METHODS
Animals and kindling procedure. Heterozygous NT-3 mutant mice were generated as described previously (11). The animals were housed under 12-h light/ 12-h dark conditions with ad libitum access to water and food. Adult male NT-31/1 (n 5 14) and NT-31/2 (n 5 13) mice, weighing 28–32 g at the start of the experiment, were used. All animals were anesthetized with Equithesin (3.0 ml/kg body wt ip) and bipolar stainless steel electrodes were implanted bilaterally into the amygdala (coordinates: toothbar at 23.3; 1.5 mm caudal to bregma; 3.0 mm lateral to midline; and 4.0 mm ventral to dura). Two weeks later, kindling stimulations (1 ms pulses, 100 Hz frequency, 1 s duration) once daily in the left amygdala were started in 10 NT-31/1 and 8 NT-31/2 mice and were continued until the animals had experienced 10 grade 5 generalized seizures. Four NT-31/1 and 5 NT-31/2 mice were nonkindled. On the first day of stimulation, the current intensity was gradually increased from 20 µA stepwise by 10 µA to determine the threshold for eliciting focal epileptiform activity (afterdischarge) of more than 5 s duration in the amygdala. This current intensity was used in subsequent stimulations. The electroencephalogram was recorded from both electrodes, and behavioral convulsions were scored blindly according to a modification of the scale of Racine (48): grade 1, facial twitches; grade 2, chewing and nodding; grade 3, forelimb clonus; grade
4, rearing, body jerks, tail upholding; and grade 5, imbalance, hind limb clonus, vocalization. Four to 12 weeks after the 10th grade 5 seizure, all stimulated mice were tested for the permanence of the kindling phenomenon. The threshold for seizure induction was determined by gradually raising the stimulation intensity similar to the initial kindling protocol, and when an afterdischarge had been induced, seizure grade and other related parameters were recorded. Two hours after the test stimulation-induced seizure, animals were anesthetized and decapitated and the brains were immediately frozen on dry ice. As assessed in cresyl violet-stained sections, the stimulating electrode was in all animals localized in the amygdaloid complex without any difference between the groups. In situ hybridization and image analysis. Unless otherwise stated, all chemicals were obtained from Sigma (Sigma-Aldrich Sweden). Cryostat sections (14 µm) were taken through the dorsal hippocampus (at a level 1.5–1.9 mm caudal to bregma) of NT-31/1 and NT-31/2 mice, which were either nonkindled and electrode-implanted (n 5 4 and n 5 5 in each group, respectively) or kindled (n 5 5, randomly selected in each group). The sections were then processed for in situ hybridization as described previously (3, 12). NT-3, BDNF, and TrkC mRNAs were detected using oligonucleotide probes complementary to nucleotides 667–717, 746–795 (12), and 1186–1236 (40) of the corresponding rat cDNA sequences, respectively. The oligonucleotide probe for full-length TrkB was complementary to nucleotides 1905–1952 of the mouse TrkB cDNA sequence (25). The probes were labeled at their 38-ends with [a-35S]dATP using terminal deoxyribonucleotidyl transferase (Amersham) to a specific activity of approximately 109 cpm/µg. After labeling, all probes were purified using Nensorb 20 columns (Dupont Medical Scandinavia). Ten millimolar dithiothreitol (DTT) was added to the labeled probes, which were then stored at 220°C. After fixation, the sections were rinsed three times during 5 min with phosphate-buffered saline (PBS), dehydrated, and air-dried. Hybridization was performed in 50% formamide, 43 SSC, 13 Denhardt’s solution, 10% dextran sulfate, 0.5 mg/ml sheared and denatured salmon sperm DNA, 1% sarkosyl (N-lauroyl sarcosine), 0.02 M phosphate buffer (PB; pH 7.0), and 0.1 M DTT using 107 cpm/ml of the respective probe. The sections were hybridized overnight at 42°C in a humidified chamber with parafilm coverslips and subsequently washed four times (15 min each) at 55°C in 13 SSC. After washing, all sections were dehydrated, air-dried, and, together with radioactive isotope standards (14C micro-scales; Amersham), exposed onto b-max X-ray film (Amersham) for 10–14 days. For cellular resolution, slides were dipped in Ilford K5 liquid photo-
KINDLING IN NT-3 MUTANT MICE
emulsion (diluted 1:1 in water), exposed for 4–6 weeks, developed in Kodak D-19, and lightly counterstained with hematoxylin–eosin. Quantification of hybridization signals was performed blindly by computerized image analysis using Image 1.57 software (Wayne Rasband, NIMH). Gray levels from the standards were used in a third degree polynomial calibration to obtain equivalent values of tissue radioactivity (nCi/g) from optical densities measured on the films. Background optical densities were subtracted from the hybridization signals. In every section, two to three measurements were made in the brain region of interest. Three sections were analyzed for each brain, and the mean value of the measurements was used for the statistical analysis. Morphological analysis. Cresyl violet-stained coronal sections (14 µm; alternate sections to those for in situ hybridization) were used to study gross morphological characteristics in the forebrain of NT-31/1 and NT-31/2 nonkindled (n 5 4 and n 5 5, respectively) and kindled (n 5 5 in each group) mice. All light microscopical analyses were performed blindly. The thickness of the dentate gyrus, CA1 and CA3 regions, and piriform and parietal cortices was measured using 103 or 403 objective. We also counted the total number of neurons in the dentate gyrus hilus and the cell numbers in 0.036-mm2 areas in the dentate granule cell layer, CA1 and CA3 pyramidal layers, basolateral amygdaloid nucleus, and piriform and parietal cortices. The analysis was carried out at comparable levels and areas in the different animals. For each region and animal, one value was obtained which represents the mean of three measurements or cell counts per section in three sections. This value was considered as one data entry in the statistical comparison between groups. Statistical evaluation. Kindling parameters in the different groups were analyzed using Student’s unpaired t test and autoradiographic and morphological data using one-way ANOVA followed by Bonferroni’s post hoc test. Student’s paired t test was performed to compare thresholds for seizure induction during kindling and test stimulations, and Kendall rank correlation was used for correlation analyses. The data are presented as means 6 SEM, and differences are considered significant at P , 0.05. RESULTS
Kindling Development Is Delayed but Maintenance Is Normal in NT-3 Mutants The behavioral expression of the different seizure grades was the same in the two strains and closely resembled that previously described in rats (48). In contrast, the development of amygdala kindling was significantly retarded in the heterozygous NT-3 mutant
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FIG. 1. Delayed kindling development in NT-31/2 mice. Bars represent mean number of amygdala stimulations to reach the different seizure grades during kindling in NT-31/1 (n 5 10) and NT-31/2 mice (n 5 8). (*) Significantly different from NT-31/1, P , 0.05; Student’s unpaired t test.
mice (Fig. 1). These mice did not exhibit the first grade 5 seizure until after 26 6 4 days of stimulation compared to 15 6 2 days in the wild-type mice (P , 0.05, Student’s unpaired t test). The fully kindled state (third grade 5 seizure) was reached after 28 6 4 days in the NT-31/2 and 17 6 2 days in the NT-31/1 mice. The deficit in the NT-3 mutants mainly reflected dampening of the progression from focal to generalized seizures. There was no significant difference between the groups in the number of stimulations required to evoke the first grade 1 or 2 focal seizures (Fig. 1). In contrast, the NT-3 mutants spent a considerably longer period of time (13 6 3 days) than wild-type mice (2 6 1 days) in grade 2 seizures. Seizure threshold, duration of afterdischarge at various stages, and latency to and duration of behavioral convulsions did not differ significantly between the groups (Table 1). In order to investigate whether the NT-3 gene mutation had influenced the permanence of the kindling phenomenon, NT-31/1 and NT-31/2 mice were left unstimulated for 4–12 weeks after having experienced 10 grade 5 seizures. When a test stimulus was subsequently given, the animals in both groups responded with a generalized seizure, indicating that the kindled state was maintained. There was no difference in seizure grade between wild-type mice and NT-3 mutants (4 6 0.9 and 4 6 0.5, respectively). The threshold to induce seizure, afterdischarge duration, and latency to and duration of behavioral convulsions were also similar in the two groups (Table 2). Furthermore, there
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TABLE 1 Seizure Characteristics during Kindling Development in NT-3 Mutant and Wild-type Mice
Groups
Seizure threshold (µA)
Afterdischarge duration at different grades (s) 1
2
3
4
Behavioral convulsions 5
Latency (s)
Duration (s)
NT-31/1
50 6 5
20 6 2
21 6 2
26 6 2
32 6 4
30 6 3
12 6 1
18 6 1
NT-31/2
45 6 6
25 6 5
26 6 4
28 6 3
33 6 4
38 6 5
14 6 1
18 6 3
Note. Latency to and duration of behavioral convulsions were measured for grade 4–5 seizures. Values represent mean 6 SEM. No significant differences were found between NT-3 mutants and wild-type mice; P . 0.05, Student’s unpaired t test (n 5 10 and n 5 8 in the NT-31/1 and NT-31/2 groups, respectively).
was no significant difference in the threshold to induce seizures during kindling as compared to that during test stimulation in either the NT-3 mutants or the wild-type animals. Basal Expression of NT-3 mRNA Is Reduced and Regulation of TrkC mRNA after Seizures Is Perturbed in NT-3 Mutants The distribution of mRNA for NT-3 in nonkindled animals, as assessed using in situ hybridization, agreed well with previous findings in the rat (12, 36), and did not differ between the mutants and the wild-type mice. Abundant NT-3 mRNA expression was only observed in the dentate granule cell layer and in the CA2 and medial CA1 pyramidal layers, suggesting that the spatial control of NT-3 mRNA expression is not perturbed in the NT-31/2 mice. We quantified NT-3 mRNA levels in the dentate granule cell layer and in the medial CA1 and CA2 pyramidal layers using computerized image analysis. In control animals, not subjected to the kindling procedure, basal expression of NT-3 mRNA in dentate granule cells was reduced by 30% in NT-31/2 compared to that in NT-31/1 mice (P , 0.05; one-way ANOVA followed by Bonferroni’s TABLE 2 Seizure Characteristics during Test Stimulation 4–12 Weeks after Kindling in NT-3 Mutant and Wild-type Mice Behavioral convulsions
Groups
Mean seizure grade
Afterdischarge duration (s)
Seizure threshold (µA)
Latency (s)
NT-31/1
4 6 0.9
31 6 1
42 6 9
22 6 2
963
NT-31/2
4 6 0.5
54 6 9
49 6 7
29 6 6
29 6 6
Duration (s)
Note. Values represent mean 6 SEM. No significant differences were found between NT-3 mutants and wild-type mice; P . 0.05, Student’s unpaired t test (n 5 5 and n 5 8 in NT-31/1 and NT-31/2 groups, respectively; five mice in the control group were excluded due to loss of the electrodes).
post hoc test; Figs. 2A and 3). In the medial CA1 and CA2 pyramidal layers also, basal NT-3 mRNA levels were slightly (18%) lower in the NT-3 mutants (not statistically significant). At 2 h after the last generalized seizure, NT-3 mRNA levels in the wild-type mice had decreased significantly (by 75%) in dentate granule cells. A similar reduction (by 73%) occurred in NT-31/2 mice. Although the mean seizure-induced NT-3 mRNA expression in NT-31/2 mice was 28% lower compared to that in wild-type animals, this difference was not statistically significant (P . 0.05; Figs. 2A and 3). No change in NT-3 mRNA levels was detected after seizures in the CA2 and medial CA1 pyramidal layers in either group (data not shown). In order to explore the possibility that the presumed low levels of NT-3 in the mutants might have influenced the expression of the TrkC receptor, we also measured TrkC mRNA levels in the dentate granule cell layer, CA1, CA2 and CA3 pyramidal layers, parietal cortex, piriform cortex, and basolateral amygdaloid nucleus. The nonkindled NT-31/2 mice did not differ from wild-type animals in basal TrkC mRNA expression (Fig. 5B). Similar to previous observations in the rat (3), the wild-type mice exhibited a marked increase in TrkC mRNA levels (to 173% of nonkindled control) at 2 h after the last generalized seizure. In contrast, no increase in TrkC mRNA expression was detected at this timepoint in the dentate granule cell layer of the NT-3 mutants (Figs. 5B and 6B). No changes of TrkC mRNA expression were found after seizures outside the dentate gyrus (data not shown). There was no significant correlation between the time from the 10th grade 5 seizure to the test stimulation (4–12 weeks) and the seizure-evoked NT-3 or TrkC mRNA expression in the different animals. Seizure-Induced Expression of BDNF and TrkB mRNAs Is Enhanced in NT-3 Mutants The presumed deficit in the NT-3 system in the mutants could hypothetically affect the expression of other neurotrophins or their receptors. We therefore analyzed levels of mRNA for BDNF and its high-affinity
97
KINDLING IN NT-3 MUTANT MICE
increased expression of BDNF mRNA after generalized seizures in all regions analyzed (ranging between 131 and 350% of control; see Figs. 2B and 4). However, the seizure-induced level of BDNF mRNA in dentate granule cells in the mutant mice was 130% higher compared to the level in kindled, wild-type animals (P , 0.05, one-way ANOVA followed by Bonferroni’s post hoc test; Figs. 2B and 4). No difference between NT-31/1 and NT-31/2 mice in BDNF mRNA expression after seizures was observed in any other brain area (data not shown). The NT-3 mutant mice did not differ from wild-type animals in basal TrkB mRNA levels (Figs. 5A and 6A). Two hours after the last generalized seizure, a significant increase in TrkB mRNA expression (to 471% of control) was observed in the dentate gyrus of NT-31/1 mice (Figs. 5A and 6A). Similar to BDNF mRNA expression, the seizure-induced TrkB mRNA levels in this region were significantly higher (38%) in NT-31/2 compared to NT-31/1 mice. There were no differences between NT-3 mutants and wild-type mice in seizureevoked TrkB mRNA levels outside the dentate gyrus (data not shown). Since there was a trend for the afterdischarge during the last seizure to be longer in the NT-3 mutants (not statistically significant, P . 0.05), we explored whether the higher expression of BDNF and TrkB mRNAs in these animals might be dependent on the duration of seizure activity. However, there was no statistically significant correlation between either the BDNF or the TrkB mRNA levels and the afterdischarge duration in the individual NT-31/1 and NT-31/2 mice. Furthermore, the seizure-evoked BDNF and TrkB mRNA levels in the different animals did not correlate with the time interval between kindling and test stimulation. Gross Anatomy Is Normal in NT-3 Mutants
FIG. 2. Reduced basal NT-3 and increased seizure-induced BDNF mRNA levels in the dentate granule cell layer of NT-31/2 mice. Levels of (A) NT-3 and (B) BDNF mRNA under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) as quantified using image analysis on in situ hybridization autoradiograms. (*) Significant difference, P , 0.05, one-way ANOVA followed by Bonferroni’s post-hoc test.
receptor, TrkB, in the dentate granule cell layer, hippocampal CA1 and CA3 regions, amygdala, and piriform and parietal cortices of nonkindled controls and in animals 2 h after the last generalized seizure. Basal expression of BDNF mRNA in NT-31/2 mice was not different from that in NT-31/1 mice in any of these regions (see Figs. 2B and 4). Both groups showed
Examination of cresyl violet-stained sections revealed no significant differences between NT-31/1 and NT-31/2 mice in the layering or thickness of the dentate gyrus, CA1 and CA3 regions, and piriform and parietal cortices. Furthermore, the total cell number in the dentate gyrus hilus and the cell numbers in 0.036mm2 areas in the dentate granule cell layer, CA1 and CA3 pyramidal layers, basolateral amygdaloid nucleus, and piriform and parietal cortices did not differ between the mutant and the wild-type mice either in nonkindled or in kindled animals (data not shown). DISCUSSION
This study shows that the rate of amygdala kindling is significantly retarded, while the persistence of the kindled state is not affected, in mice heterozygous for a deletion of the NT-3 gene. Furthermore, in these ani-
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FIG. 3. Examples of in situ hybridization autoradiograms demonstrating the levels of NT-3 mRNA expression under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in the hippocampus of NT-31/2 and NT-31/1 mice.
mals the seizure-evoked increases in BDNF and TrkB mRNA expression in dentate granule cells are enhanced compared to those in wild-type mice. The present findings suggest that endogenous NT-3 levels can influence seizure development during epileptogenesis and that a deficit in the function of this neurotrophin may lead to changes in gene regulation of another neurotrophin, BDNF, and its high-affinity receptor, TrkB. Previous in situ hybridization studies (9, 21, 41) and measurements of NGF (4), BDNF (46), and TrkB protein (41) content indicate that basal and seizureinduced expression of neurotrophin and Trk mRNAs to a large extent reflects the level of the corresponding proteins. We observed that under basal conditions, NT-3 mRNA expression was reduced by 30% in the dentate granule cell layer of NT-31/2 compared to NT-31/1 mice. Also at 2 h after a generalized seizure, NT-3 mRNA levels seemed to be lower in the mutants, but this difference was not statistically significant. The reduction of NT-3 mRNA expression after brain insults had not reached its maximum at 2 h (32), and the difference between NT-3 mutants and wild-type animals may, therefore, be more pronounced at later timepoints. In any case, since the seizure-evoked increase in TrkC mRNA expression detected in NT-31/1 mice was not observed in the mutants, the present results suggest that the NT-3 system in the dentate granule cell layer of NT-31/2 mice is impaired both under basal conditions and after seizures. We could not detect any significant hybridization signal outside the hippocampus in the two strains, which is in agreement with previous findings in the
adult rat (12). However, RNA blot analysis has revealed a weak signal for NT-3 in cerebral cortical tissue (10), and with ELISA a relatively high level of NT-3 protein has been detected in the piriform cortex and low levels in other cortical areas (58). These data suggest that NT-3 may influence the function of the cerebral cortex also in the adult, in particular, since the TrkC receptor is abundantly expressed in this region (40). Given the effects of NT-3 on excitatory synaptic transmission in cultured cortical neurons (24), it cannot be excluded that a reduction of basal and seizure-induced levels of NT-3 protein in the cerebral cortex of the NT-31/2 mice might have contributed to the observed deficit in kindling development. It seems highly warranted to explore this possibility by measurement of NT-3 protein levels in the different CNS regions of the mutants. The effect of NT-3 on seizure development during kindling epileptogenesis might be due to a direct action of this neurotrophin on synaptic transmission. It has been shown that NT-3 enhances acetylcholine release at the Xenopus neuromuscular junction (34) and that direct injection of NT-3 into the hippocampus gives rise to epileptiform and theta-like activities, which can be blocked by the muscarinic receptor antagonist, scopolamine (5). Glutamatergic transmission at the Schaffer collateral-CA1 synapses is potentiated for 2 to 3 h after application of NT-3 to hippocampal slices (23). Furthermore, NT-3 specifically enhances impulse activity of cortical neurons in culture and induces synchronization of excitatory synaptic currents (24), probably through reduction of GABAergic synaptic transmission. The presumed reduced NT-3 protein levels in the mutant mice might lead to less inhibition of GABAergic
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99
FIG. 4. Examples of in situ hybridization autoradiograms (A) and bright-field photomicrographs (B) depicting BDNF mRNA levels in the dentate granule cell layer under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in NT-31/2 and NT-31/1 mice. Bar, 20 µm.
100
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FIG. 5. Perturbed seizure-induced TrkB and TrkC mRNA expression in the dentate granule cell layer of NT-31/2 mice. Levels of (A) TrkB and (B) TrkC mRNA under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in NT-31/2 and NT-31/1 mice as quantified using image analysis on in situ hybridization autoradiograms. (*) Significant difference, P , 0.05, one-way ANOVA followed by Bonferroni’s post-hoc test.
transmission and a subsequent reduction of neuronal activity and synchronization, resulting in delayed epileptogenesis. In support of this hypothesis, synchronization of neuronal activity appears to be crucial for generating epileptic events (35, 42), and reduction of excitatory glutamatergic (39, 43) or increase in inhibi-
tory GABAergic transmission (22, 53, 54, 64) leads to suppressed seizure development. Another possibility is that the retardation of kindling rate in the mutants is due to reduced levels of NT-3 during CNS development, causing abnormalities in the formation of anatomical connections or brain regions important for seizure generalization. NT-3 has been shown to promote neuronal differentiation of cortical and hippocampal precursor cells (17, 63) and to support survival and differentiation of noradrenergic locus coeruleus neurons (1, 15). Arguing against a morphological deficit in the NT-31/2 mutants, the spread of seizure activity from the stimulated to the nonstimulated amygdala and the expression of behavioral convulsions were unchanged in these animals compared to wildtype mice. Furthermore, a dysfunction of the noradrenergic system should, most likely, have facilitated and not delayed kindling epileptogenesis (37). Finally, we did not detect any morphological abnormalities in the NT-31/2 mice, which is in agreement with previous observations in the forebrain of NT-32/2 mice (11, 25). The enhancement of the seizure-evoked increase in BDNF and TrkB mRNA expression in NT-31/2 mice was confined to dentate granule cells. BDNF and NT-3 probably act on the same dentate granule cells, most of which express both the TrkB and the TrkC receptor (28). It may be hypothesized that BDNF and NT-3 mechanisms in these cells are linked and that a deficit in the activation of the TrkC receptor causes an upregulation of BDNF and TrkB synthesis. Supporting the idea that one neurotrophin may influence the regulation of another neurotrophin, BDNF and NT-4 (which both act on the TrkB receptor) increase NT-3 mRNA expression in hippocampal neurons (30). Hypothetically, the balance between the BDNF and the NT-3 systems after seizures, suggested by our data, may be important for the fine tuning of synaptic efficacy in the hippocampus. However, the present findings suggest that the presumed increase in the function of the BDNF system after seizures is insufficient to preserve normal excitability and kindling development in NT-3 mutants. In our previous study (27), heterozygous BDNF knockout mice, which had significantly reduced BDNF mRNA levels in dentate granule cells but exhibited no perturbation of NT-3 mRNA regulation after seizures, showed marked inhibition of kindling epileptogenesis. The less pronounced retardation of kindling development in the NT-31/2 compared to the BDNF1/2 mice could, at least partly, be attributable to the enhanced seizure-induced BDNF and TrkB synthesis in the dentate granule cells of the NT-3 mutants. In conclusion, the present study provides evidence that NT-3, similar to what has recently been reported for NGF (16, 61) and BDNF (27), can influence kindling epileptogenesis. For NGF, this effect may be exerted through the induction of mossy fiber sprouting (49, 61),
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FIG. 6. Examples of in situ hybridization autoradiograms demonstrating TrkB (A) and TrkC (B) mRNA expression under basal conditions (nonkindled) and at 2 h after the last generalized seizure (kindled) in the hippocampus of NT-31/2 and NT-31/1 mice.
whereas it seems more likely that NT-3 and BDNF can act via a direct effect on synaptic transmission. Kindling is an animal model of the most common type of epilepsy in adult humans, complex partial seizures (38), and kindling-like processes probably contribute to epileptogenesis in patients. Our data suggest that the downregulation of NT-3 synthesis after seizures and other brain insults, such as traumatic injury, can act to dampen the development of epilepsy. Furthermore, they suggest that strategies
leading to the attenuation of neurotrophin levels after these insults might counteract epileptogenesis in humans. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council, the Swedish Society for Medical Research, the Medical Faculty, University of Lund, the Royal Physiographic Society, and the Wiberg, Kock, Elsa Schmitz, and Elsa & Thorsten
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Segerfalk foundations. In addition, P.E. was supported by the Swedish Cancer Society and the Augusta and Petrus Hedlunds foundation. M.K. and Z.K. were partly supported by funds from the I. Beritashvili Institute of Physiology, Georgian Academy of Sciences. We are grateful to Marie Lundin for secretarial work and to Agneta Othberg for skillful technical assistance.
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