Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

Abstract

The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca(2+)) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities.

Fig. 1.

Treatment of embryos and larvae with the GABA pathway inhibitor pentylenetetrazole induces convulsions that are characterised by increased locomotor activity and calcium influx into muscle cells. (A) 4 dpf larvae were exposed to 1.25, 2.5, 5, 10, 20, 40 or 80 mM PTZ for 10 minutes, then movements were recorded over the next 10 minutes. Values are given as means (n=4); error bars indicate s.e.m. ***Significantly different (P<0.001) from untreated embryos, using one-way ANOVA with Dunnett’s post-test. Representative traces of movements after exposure to each PTZ concentration lie beneath each corresponding value for PTZ concentration in the histogram. Black, green and red traces indicate swimming speeds of 0–1.5, 1.5–6 and >6 mm/second, respectively. (B) In situ hybridisation analysis reveals strong expression of GABA_A receptor component genes gabra1 and gabrg2 throughout the brain of 50 hpf embryos. (C) Induction of convulsions in embryos by exposure to PTZ. Panel 1: representative traces of locomotor activity of 48 hpf embryos untreated (−PTZ) and exposed to 20 mM PTZ (+PTZ). Black, green and red traces indicate swimming speeds of 0–1.5, 1.5–6 and >6 mm/second, respectively. Panels 2, 3 and 4: live confocal imaging of GCaMP-3 fluorescence in the muscle of a 50 hpf zebrafish embryo transiently expressing a mylz2:GCaMP3 reporter transgene. Images are taken from a movie after 5 minutes of exposure to 20 mM PTZ, before (Panel 2), during (Panel 3) and after (Panel 4) a single convulsion. Panel 5: graph of GCaMP-3 fluorescence from the embryo in Panels 2–4, over a period in which two consecutive convulsions occurred.

Fig. 2.

Pentylenetetrazole and picrotoxin treatments induce expression of the synaptic-activity-regulated gene fos in 2-day-old zebrafish embryos, and this expression is suppressed by the anticonvulsant drug VPA. (A) Treatment of 50 hpf embryos with the GABA_A receptor inhibitors PTZ (20 mM) or picrotoxin (300 μM) caused rapid induction of fos expression in the forebrain within 30 minutes of addition of drug, which was increased at 60 and 90 minutes after initial addition. Treatment of 50 hpf embryos with PTZ (20 mM) also induced strong fos expression in the trunk muscle after 30, 60 and 90 minutes of treatment, whereas treatment with picrotoxin (300 μM) only induced detectable fos expression after 90 minutes of treatment. (B) PTZ-induced expression of fos in the developing CNS is greatest in the ventral telencephalon (subpallium; arrows) and ventral diencephalon (arrowheads) within the forebrain of 50 hpf embryos. In the ventral diencephalon, the fos expression domain induced by PTZ encompasses the preoptic area, posterior tuberculum and hypothalamus. (C) Treatment of embryos with the anticonvulsant VPA suppresses PTZ- and picrotoxin-induced expression of fos in the ventral telencephalon and ventral diencephalon. 50 hpf embryos were incubated for 60 minutes in E3 medium containing 1 mM VPA or E3 medium only (no VPA), after which either 20 mM PTZ (+PTZ) or 300 μM picrotoxin (+Picrotoxin) was added and embryos were incubated for a further 90 minutes. Embryos were then fixed and analysed for expression of fos transcripts.

Fig. 3.

Increased expression of CNS-specific genes accompanies transcriptional activation of fos by pentylenetetrazole. Exposure of 50 hpf embryos to 20 mM PTZ induced expression of genes encoding the transcription factor Npas4 and the secreted neurotrophic factor Bdnf in the ventral forebrain, both of which are known to be synaptic-activity-regulated, within 90 minutes of exposure. Expression of fos in the CNS was strongest in the regions of the forebrain that also expressed npas4 and bdnf. Increased expression of pyya, encoding a homologue of Neuropeptide Y, was also observed at multiple locations within the CNS, most prominently within the posterior hindbrain. Left panels, embryos incubated in E3 medium only; right panels, embryos incubated in E3 medium containing 20 mM PTZ for 90 minutes.

Fig. 4.

Identification of known neuroactive compounds with anticonvulsant activity in the zebrafish embryo. Known inhibitors of neural activity were identified within the Microsource Spectrum Collection on the basis of their concentration-dependent suppression of PTZ-induced fos expression and inhibition of PTZ-induced locomotor activity. Compounds were administered to 50 hpf embryos for 90 minutes in 96-well plates, at a final concentration of 0.9, 2.7, 8.2, 24.7 or 74.1 μM (as indicated), then 20 mM PTZ was added and embryos were incubated for a further 60 minutes, before being fixed and analysed for expression of fos. In parallel, 4-day-old zebrafish larvae were exposed to the same range of compound concentrations in 48-well plates for 90 minutes, then PTZ was added and larvae were incubated for a further 10 minutes. 48-well plates were then transferred to the Viewpoint Zebrabox for live tracking of locomotor activity over a 10 minute period. Black, green and red traces indicate swimming speeds of 0–1.5, 1.5–6 and >6 mm/second, respectively. (A) Ethinylestradiol; (B) allopregnanolone; (C) nimodipine; (D) nitrendipine; (E) methiothepin; (F) pimozide. (G) The anticonvulsant activity of the positive control compound VPA is shown, for comparison.

Fig. 5.

Identification of compounds with anticonvulsant activity in the zebrafish embryo. Previously unknown inhibitors of neural activity were identified within the Microsource Spectrum Collection, on the basis of their concentration-dependent suppression of PTZ-induced fos expression and inhibition of PTZ-induced locomotor activity. Compounds were administered to embryos for 90 minutes in 96-well plates, at a final concentration of 0.9, 2.7, 8.2, 24.7 or 74.1 μM (as indicated), then 20 mM PTZ was added, and embryos were then incubated for a further 60 minutes before being fixed and analysed for expression of fos. In parallel, 4-day-old zebrafish larvae were exposed to the same range of compound concentrations in 48-well plates for 90 minutes, then PTZ was added and larvae were incubated for a further 10 minutes. 48-well plates were then transferred to the Viewpoint Zebrabox for live tracking of locomotor activity over a 10 minute period. Black, green and red traces indicate swimming speeds of 0–1.5, 1.5–6 and >6 mm/second, respectively. (A) Sulconazole; (B) suloctidil; (C) nerolidol; (D) dioxybenzone; (E) hexylresorcinol; (F) retinyl acetate.

Fig. 6.

Quantitative comparison of the concentration-dependent effects of identified anticonvulsants on PTZ-induced locomotor activity of 4-day-old zebrafish larvae. Compounds were administered to larvae for 90 minutes in 48-well plates, at a final concentration of 2.7, 8.2 or 24. μM, then 20 mM PTZ was added and larvae were then incubated for a further 10 minutes before being transferred to the Viewpoint Zebrabox for live tracking of locomotor activity over a 10 minute period. For each test compound, n=9; for VPA controls (V), n=71; for PTZ controls (P), n=108; for E3-only controls (E), n=96. Error bars show s.e.m. Asterisks indicate values that are significantly different from the value for PTZ-treated embryos (P), using a one-way ANOVA analysis with Dunnett’s post-test. Level of statistical significance is indicated: ***P<0.001; **P<0.01; *P<0.05.