Brain-derived neurotrophic factor induces hyperexcitable reentrant circuits in the dentate gyrus

Abstract

Aberrant sprouting and synaptic reorganization of the mossy fiber (MF) axons are commonly found in the hippocampus of temporal lobe epilepsy patients and result in the formation of excitatory feedback loops in the dentate gyrus, a putative cellular basis for recurrent epileptic seizures. Using ex vivo hippocampal cultures, we show that prolonged hyperactivity induces MF sprouting and the resultant network reorganizations and that brain-derived neurotrophic factor (BDNF) is necessary and sufficient to evoke these pathogenic plasticities. Hyperexcitation induced an upregulation of BDNF protein expression in the MF pathway, an effect mediated by L-type Ca2+ channels. The neurotrophin receptor tyrosine kinase (Trk)B inhibitor K252a or function-blocking anti-BDNF antibody prevented hyperactivity-induced MF sprouting. Even under blockade of neural activity, local application of BDNF to the hilus, but not other subregions, was capable of initiating MF axonal remodeling, eventually leading to dentate hyperexcitability. Transfecting granule cells with dominant-negative TrkB prevented axonal branching. Thus, excessive activation of L-type Ca2+ channels causes granule cells to express BDNF, and extracellularly released BDNF stimulates TrkB receptors present on the hilar segment of the MFs to induce axonal branching, which may establish hyperexcitable dentate circuits.

Figure 7.

BDNF promotes the formation of axonal protrusions of granule cells. A, Confocal images of isolated granule cells stained with rhodamine phalloidin (red), anti-tau-1 (green), and anti-MAP-2 (blue) after 24 hr treatment with (right) or without (left) 100 ng/ml BDNF. In the following analyses, we regarded the longest, tau-1-positive, MAP-2-nevative neurite as an axon and measured the density of phalloidin-positive protrusions (arrows) along the axon length. B, Dose-dependent effect of BDNF on the density of axonal protrusions. BDNF was added in the media at day 4 in culture and continuously present for 24 hr. Observation was performed at day 5. C, Axonal protrusions were counted in cultures treated for 24 hr with or without 10-100 ng/ml BDNF in the absence or presence of 300 nm K252a and 10 μm nicardipine. ^**p < 0.01 versus control; ^##p < 0.01 versus BDNF; Tukey's test after ANOVA. Data are means ± SEM of 162-263 axonal segments in eight different series of experiments.

Figure 8.

BDNF directly acts on granule cells to induce axonal sprouting. A, Confocal images of lacZ- or dominant-negative TrkB (dnTrkB)-transfected neurons. Granule cells were transfected with cDNAs (IRES-GFP) at day 4 in vitro. Twenty-four hours after transfection, cultures were exposed to 10 ng/ml BDNF for 24 hr and stained with rhodamine phalloidin (red) and anti-MAP-2 (blue). Transfection with lacZ was used as a negative control for gene expression. B, Quantitative analysis of axonal protrusions. Data of “not transfected” were derived from neighboring untransfected neurons in the same batches. ^**p < 0.01; Tukey's test after ANOVA. Data are means ± SEM of 116-254 axon segments in eight different series of cultures.

Figure 1.

Endogenous BDNF mediates activity-dependent MF reorganization. A, Representative Timm staining of hippocampal slice cultures maintained for 10 d in the absence (left) and presence of 50 μm picrotoxin (middle) and a combination of 50 μm picrotoxin and 1.6 μg/ml function-blocking anti-BDNF antibody (right). MF heterotopias (arrows) in the inner molecular layer (ml) of picrotoxin-treated hippocampus were blocked by cotreatment with anti-BDNF antibody. B, Measurement of Timm grain intensity. In an image taken with a 20× objective, atleast five 20 × 20 μm cursors were put in each stratum, i.e., hilus, and inner molecular layer (ml), and subicular area (subic) located immediately outside the hippocampal fissure(hf), and the mean signal intensity(I) within the cursors was measured at 8 bit resolution. The I values were averaged for each stratum (<I_hilus>, <I_ml>, and <I_subic>). Timm grain intensity was defined as <I_hilus>/<I_subic> and <I_ml>/<I_subic>. C, Timm grain intensities of the dentate hilus and molecular layer were quantified in control slices and slices that received 10 d treatment with 50 μm picrotoxin in the absence or presence of 1 μm tetrodotoxin or 10 μm nicardipine. All experiments were repeated with at least four different experiments, producing similar results, with no significant variation from experiment to experiment (Ftest). ^**p< 0.01; Tukey's test after ANOVA. Data are means ± SD of each of 4-16 slices. D, Same analysis performed with slices cotreated with 50 μm picrotoxin and 300 nm K252a or 1.6 μg/ml anti-BDNF antibody.

Figure 2.

Lamina-specific induction of BDNF protein in hyper excited hippocampus. A, ELISA-based quantification of changes in BDNF expression in hippocampal slices cultivated in the absence and presence of 50 μm picrotoxin. B, BDNF was quantified in slices exposed to 50 μm picrotoxin for 2 d in the absence and presence of 1 μm tetrodotoxin and 10 μm nicardipine. ^*p < 0.05; ^**p < 0.01 versus control; ^##p < 0.01 versus picrotoxin; Tukey's test after ANOVA. Data are means ± SD of 4-8 slices. C, Immunohistochemical staining for BDNF in control and picrotoxin-treated (50 μm for 10 d) slices. BDNF expression was selectively induced in the stratum lucidum (sl), hilus and molecular layer (ml), but not the stratum oriens (so), pyramidale (sp), radiatum (sr), or granulosum (sg).

Figure 3.

Late onset of hyperexcitation-induced mossy fiber sprouting. A, Representative Timm images around the crest of dentate gyrus including the dentate hilus (hilus), stratum granulosum and innermolecular layer(ml) of slices cultured in the presence of 50 μm picrotoxin for 1, 5, and 10 d. Arrows indicate sprouted MFs. B, Time course of occurrence of mossy fiber sprouting after picrotoxin application. Timm grain intensities of the dentate hilus (circles) and molecular layer (squares) were quantified in the absence (open symbols) or presence (closed symbols) of 50 μm picrotoxin at 1-10 d in vitro. Hyperexcitation did not induce mossy fiber sprouting until at least 5 d after application. ^**p < 0.01 versus control; Tukey's test after ANOVA. Data are means ± SD of each of 5-8 slices obtained from four independent experiments.

Figure 4.

Exogenous application of BDNF to the hilus, but not other subregions, induces activity-independent MF sprouting. A, Timm images of hippocampal slices with their hilar regions carrying PBS-including (left) and BDNF-including beads (right) for 10 d. Ectopic MF terminals (arrows) were found in the molecular layer (ml) of the BDNF bead-bearing slice. B, Timm grain intensities of the dentate hilus and molecular layer were quantified in slices in which BDNF-containing beads were put on their hilus, molecular layer, stratum lucidum, or stratum radiatum for 10 d. In the data of “bath application”, 100 ng/ml BDNF was directly added to culture medium without beads, and the medium was replaced with fresh ones containing 100 ng/ml BDNF every 24 hr until day 10. C, Same paradigm of analysis as shown in B, from bead-carrying slices treated for 10 d with 300 nm K252a, 1.6 μg/ml anti-BDNF antibody, 10 μm CNQX, 100 μm AP-5, 1 μm tetrodotoxin, or 10 μm nicardipine. ^**p < 0.01 versus control or no bead; ^##p < 0.01 versus BDNF bead; Tukey's test after ANOVA. Data are means ± SD of each of 4-12 slices obtained from four independent experiments.

Figure 5.

Transient exposure to BDNF is enough to induce MF sprouting. A, Experimental procedure. A control or BDNF-containing bead was put on the dentate hilus at day 0 in vitro. To block the action of BDNF, K252a was applied at day 0-9 in vitro and continuously present in the culture medium until day 10 when sprouting was assessed by Timm staining. B, Timm grain intensities of the dentate hilus (circles) and molecular layer (squares) were quantified in control slices (open symbols) and slices that received 10-day treatment with BDNF-including beads and were cultured in the absence (closed symbols) or presence of 300 nm K252a (gray symbols). Exposure to BDNF for as little as 1 d was capable of inducing MF sprouting. ^**p < 0.01; Tukey's test after ANOVA. Data are means ± SD of each 5-11 slices obtained from four independent experiments.

Figure 6.

Granule cells display prolonged epileptiform activity after chronic BDNF treatment. A, Schematic draw of positions of stimulating (Stim.) and patch-clamp recording (Rec.) electrodes. Response of a granule cell present in the stratum granulosum (sg) to a single-pulse stimulus of the dentate hilus was monitored with whole-cell current clamp recording. Recording was performed with a granule cell located at least 350 μm apart from a stimulating electrode, and the intensity of filed stimulation were carefully adjusted to generate no antidromic activation in the recorded neurons, which was checked by application of tetrodotoxin at the end of experiments. B-E, Each trace on the bottom indicates a typical intracellular response to local stimulation of the hilus, recorded from the corresponding neurobiotin-filled, camera lucida-reconstructed granule cell (top) in a control (B) or 50 μm picrotoxin-treated slice (C), or a slice carrying a control (D) or BDNF-including (E) bead in the hilus. Hilar stimulation (arrowheads) caused paroxysmal activation of the granule cells in picrotoxin- or BDNF-treated slices, in which MF collaterals often invaded the stratum granulosum (sg) and even the molecular layer (ml), as indicated by the arrows.