H.L., S.-Y.S., W.-N.C. and T.-F.F. conceived and designed the experiments. G.-H.L., T.-T.K. and H.-C.D. performed the experiments. G.-H.L. and T.-F.F. analyzed the data. M.K.S. provided ginkgotoxin and ginkgotoxin phosphate.
FUNDING
This work was supported by the National Science Council, Taiwan [grant number NSC 99-2320-B-006-013-MY3] and the Program for Promoting Academic Key Performance Indicator (KPI) Excellence of Universities, National Cheng Kung University (NCKU) [grant number D100-35B41].
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Figure Legends
Fig. 1. Seizure-like behavior observed in zebrafish larvae exposed to ginkgotoxin.
Zebrafish larvae at 5 dpf were incubated in water containing ginkgotoxin of various concentrations for 1 hour (upper) or 2 hours (lower) before observing under a microscope. Ginkgotoxin-exposed larvae were categorized into three stages based on the characteristics of their swimming behavior: stage 0, no or low swimming activity;
stage I, mildly increased swimming activity; stage II, whole body convulsion and misshaped. There were a total of ten larvae in each group.
Fig. 2. Schedule for treatment with ginkgotoxin and anti-convulsing compounds and data acquisition. Embryos at three different stages were collected separately into three groups, namely 3-dpf/2-hr, 5-dpf/2-hr and 6- hpf/66-hr. The embryos in each group were divided into three subgroups (1, 2, 3) and treated with ginkgotoxin and AEDs following three different strategies. Subgroup 1 was treated with ginkgotoxin (GT) only for the duration indicated by solid arrow before video-recording for data acquisition. Subgroup 2 was treated simultaneously with both ginkgotoxin and rescuing compounds or AED for the duration indicated by dashed arrow before data acquisition. Subgroup 3 was treated with ginkgotoxin first for the duration indicated by the solid arrow. Then, the ginkgotoxin was replaced with rescuing compounds or AED and larvae were incubated for additional 3 hours before data acquisition. 3-dpf/2-hr refers to 3-dpf embryos incubated in ginkgotoxin for 2 hours. 5-3-dpf/2-hr refers to 5-dpf embryos incubated in ginkgotoxin for 2 hours. hpf/6hr refers to 6-hpf embryos incubated in ginkgotoxin for 66 hours.
Fig. 3. Quantitative analysis of behavioral seizures induced by ginkgotoxin exposure and of response to AEDs. Embryos at indicated stages (3 dpf, 5 dpf or 6 hpf) were co-treated with both ginkgotoxin (GT) and anti-convulsing compounds simultaneously for the indicated time duration before video-recording (subgroup 2).
(A)Sample locomotion tracking of 1.5 minutes are shown for individual larva at the end of treatment. (B)The traveling distance and velocity of larval displacement were analyzed. At least ten larvae were recorded for each group. Bars show mean + s.e.m.
**P<0.0001.
Fig. 4. PLP-requiring folate-mediated one-carbon pools affected byginkgotoxin exposure. Zebrafish embryos at 6 hpf were raised in fish water (control) or water containing 500 M ginkgotoxin (GT) until 3 dpf and then harvested for folate content analysis. Total folate was measured with a microbial assay, whereas individual folate species were measured with HPLC. No significant differences were observed in folate content between the ginkgotoxin-treated group (gray bar) and control group (black bar). Bars show mean + s.e.m. THF, tetrahydrofolate.
Fig. 5. Ginkgotoxin does not affect the development and appearance of zebrafish larvae. Zebrafish embryos were raised in fish water (control) or water containing 0.5 mM ginkgotoxin (GT) from 6 hpf to 72 hpf. (A-D)The lateral (A,C) and dorsal (B,D) views of embryos revealed no observable morphological differences in larval size and appearance between control and ginkgotoxin-treated groups. (E-H)The H&E-stained transverse cryosections of larval head regions and anterior trunk are oriented dorsal (top) to ventral (bottom) (E, F). Histochemical examination of the myotomes and notochord of embryos revealed no obvious difference between the control and
ginkgotoxin-treated groups despite the variation in their shape (G,H). (I-P)The development of embryos were characterized at 24 hpf with in-situ hybridization using probes specific for somatic muscle (myo D, caudal lateral trunk views), rhombomere (krox20, lateral view with anterior to the left) and glutamatergic neurons (vglut2.1 and vglut2.2, dorsal view). (Q,R)The posterior lateral line formation was characterized at 72 hpf by staining with DASPEI. m, myotome; n, notochord; e, eyes; d, diencephalon;
t, tectum; s, somite; r3, rhombomere 3; r5, rhombomere 5.
Fig. 6. Ginkgotoxin-induced malformation of optic stalk and spinal cord neuron was partially reversed by PLP or GABA supplementation. Zebrafish embryos were raised in fish water (control) or water containing 500 M ginkgotoxin added at 6
hpf (GT). (A,B) The development of embryos were characterized at 24 hpf using in-situ hybridization with pax2.1 probe (lateral view with anterior to the left).
Development of ostic stalk and spinal cord neuron were interfered with in the ginkgotoxin-treated group (B) and the effect was reversed in the presence of 0.5 mM PLP (C) or 0.5 mM GABA (D). Adding PLP alone did not cause observable changes in embryos (E), whereas adding GABA alone to the water impeded the development of pax2.1- expressing tissues, especially optic stalk, otic vesicle and spinal cord neuron (F). (G,H)Confocal microscopy images of the nortochord in the trunk of Tg(alx:GFP) transgenic embryos at 3 dpf. OS, optic stalk; MHB, midbrain-hindbrain boundary; OV, otic vesicle; SPN, spinal cord neurons; PD, pronephric duct. Scale bar:
8 m.
Fig. 7. Primidone relieved the larval seizure-like behavior induced by ginkgotoxin exposure but not by PTZ treatment. Zebrafish larvae at 3 dpf were
incubated in water containing convulsing agents with or without the presence of primidone for 2 hours before video recording and analyzed for the stage distribution (left) and total distance of larval displacement (right). The larval behavior was categorized into three stages on the basis of the characteristics of their swimming patterns: stage 0, no or low swimming activity; stage I, mildly increased swimming activity; stage II, whole body convulsion, erupting swimming and misshaped. There were at least ten larvae included for each group. Bars show mean + s.e.m.
**P<0.0001.
Fig. 8. Speculated mechanisms underlying ginkgotoxin-induced seizures.
Ginkgotoxin and ginkgotoxin phosphate inhibit pyridoxal kinase (PL kinase) for substrates pyridoxal, pyridoxamine and pyridoxine, resulting in decreased levels of PLP and pyridoxine-5_-phosphate (PNP). Inhibition of PL conversion to PLP directly decreases the amount of PLP formed by this pathway. The decrease in formation of PNP reduces PLP formation by pyridoxine phosphate oxidase. There is also some inhibition of pyridoxine phosphate oxidase by ginkgotoxin phosphate (Salamon et al., 2009). A lower in vivo PLP concentration reduces the conversion of apo-GAD to holo-GAD (GAD to GADPLP). Ginkgotoxin phosphate might also compete with PLP for binding to apo-GAD. Both mechanisms result in decreased intracellular GAD activity and GABA formation, leading to the occurrence of seizures. PN, pyridoxine;
PM, pyridoxamine; PNP, pyridoxine-5_-phosphate; PMP, pyridoxamine-5_-phosphate; PL, pyridoxal; GT, ginkgotoxin.