Pharmacological Analysis of the Anti-epileptic Mechanisms of Fenfluramine in scn1a Mutant Zebrafish

Abstract

Dravet syndrome (DS) is a genetic encephalopathy that is characterized by severe seizures and prominent co-morbidities (e.g., physical, intellectual disabilities). More than 85% of the DS patients carry an SCN1A mutation (sodium channel, voltage gated, type I alpha subunit). Although numerous anti-epileptic drugs have entered the market since 1990, these drugs often fail to adequately control seizures in DS patients. Nonetheless, current clinical data shows significant seizure reduction in DS patients treated with the serotonergic (5-hydroxytryptamine, 5-HT) drug fenfluramine (FA). Recent preclinical research confirmed the anti-epileptiform activity of FA in homozygous scn1a mutant zebrafish larvae that mimic DS well. Here we explored the anti-epileptiform mechanisms of FA by investigating whether selective agonists/antagonists of specific receptor subtypes were able to counteract the FA-induced inhibition of seizures and abnormal brain discharges observed in the scn1a mutants. We show that antagonists of 5-HT_1D and 5-HT_2C receptor subtypes were able to do so (LY 310762 and SB 242084, respectively), but notably, a 5-HT_2A-antagonist (ketanserin) was not. In addition, exploring further the mechanism of action of FA beyond its serotonergic profile, we found that the anti-epileptiform brain activity of FA was significantly abolished when it was administered in combination with a σ₁-agonist (PRE 084). Our study therefore provides the first evidence of an involvement of the σ₁ receptor in the mechanism of FA. We further show that the level of some neurotransmitters [i.e., dopamine and noradrenaline (NAD)] in head homogenates was altered after FA treatment, whereas γ-aminobutyric acid (GABA) and glutamate levels were not. Of interest, NAD-decreasing drugs have been employed successfully in the treatment of neurological diseases; including epilepsy and this effect could contribute to the therapeutic effect of the compound. In summary, we hypothesize that the anti-epileptiform activity of FA not only originates from its 5-HT_1D- and 5-HT_2C-agonism, but likely also from its ability to block σ₁ receptors. These findings will help in better understanding the pharmacological profile of compounds that is critical for their applicability in the treatment of DS and possibly also other drug-resistant epilepsies.

Keywords: Dravet syndrome; GABA; Zebrafish; epilepsy; glutamate; monoamines; pharmacological modulation; sigma.

FIGURE 1

Experimental workflow. Selection of compounds based on hypothesized mechanisms of FA: stimulation (agonist, left side) or inhibition (antagonist, right side) of specific receptors. Light gray boxes show single treatment and dark gray boxes represent combinatorial treatment with FA.

FIGURE 2

Activity profile of the selective beta₂-antagonist (β₂), sigma₁-antagonist (σ₁), sigma₂-antagonist (σ₂), subtype selective 5-HT-agonists (1D, 2A, 2C) and triple treatment with anti-epileptiform compounds [σ₁-antagonist and subtype selective 5-HT-agonists (1D and 2C)] (locomotor behavioral assays). Locomotor activity was normalized against VHC-treated scn1a mutant larvae and displayed as a percentage ± SD. (A1) Normalized locomotor activity of treated scn1a mutant larvae (-/-). (A2) Normalized locomotor activity of treated WT larvae (+/+). (B) Percentage of scn1a mutant larvae with epileptiform activity below (white area) or above the threshold value (black area). Treatment with the selective β₂-antagonist, σ₁-antagonist, the subtype selective 5-HT-agonists (1D, 2A, 2C) and triple treatment with anti-epileptiform compounds [σ₁-antagonist and subtype selective 5-HT-agonists (1D and 2C)] induced anti-epileptiform locomotor activity. A statistical difference is indicated by: ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. VHC-treated controls. n = 16–30 ZF larvae for all experimental conditions.

FIGURE 3

Activity profile of the selective beta₂-antagonist (β₂), sigma₁-antagonist (σ₁), sigma₂-antagonist (σ₂), subtype selective 5-HT-agonists (1D, 2A, 2C) and triple treatment with anti-epileptiform compounds [σ₁-antagonist and subtype selective 5-HT-agonists (1D and 2C)] (forebrain LFP recordings). Quantification of brain recordings of VHC-treated WT larvae [VHC(+/+), n = 42], VHC-treated scn1a mutant larvae [VHC(-/-), n = 41] and compound-treated scn1a mutant larvae [compound(-/-)]. (A) Frequency of epileptiform events during 10 min recording. (B) Mean cumulative duration of epileptiform events during 10 min recording. Treatment with the selective β₂-antagonist (n = 19) or the σ₂-antagonist (n = 14) did not affect epileptiform brain activity in scn1a mutant larvae. In contrast, the σ₁-antagonist (n = 20), 5-HT_1D-agonist (n = 10), 5-HT_2A-agonist (n = 10), the 5-HT_2C-agonist (n = 14) and triple treatment with anti-epileptiform compounds [σ₁-antagonist and subtype selective 5-HT-agonists (1D and 2C); n = 11] significantly reduced the frequency and the mean cumulative duration of epileptiform brain activity in scn1a mutant larvae. A statistical difference is indicated by: ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 vs. VHC-treated scn1a mutant larvae. (C) Visualization of representative electrograms of 7 dpf ZF larvae: VHC-treated WT larva [VHC (+/+)], VHC-treated scn1a mutant larva [VHC (-/-)]; β₂-antagonist-treated scn1a mutant larva [β₂-antagonist (-/-)]; triple treatment of scn1a mutant larva with anti-epileptiform compounds [σ₁-antagonist+5-HT_1D&2C-agonists (-/-)]; scale bars: 1 mV, 30 s.

FIGURE 4

Activity profile of FA [single treatment with FA and combinatorial treatment of FA with the sigma₁-agonist (σ₁) or subtype selective 5-HT-antagonists (1D, 2A, 2C, 2B) or triple treatment of epileptiform compounds [σ₁-agonist and subtype selective 5-HT-antagonists (1D and 2C)] (locomotor behavioral assays)]. Locomotor activity was normalized against VHC-treated scn1a mutant larvae (-/-) and displayed as a percentage ± SD. (A1) Normalized locomotor activity of treated scn1a mutant larvae (-/-). (A2) Normalized locomotor activity of treated WT larvae (+/+). (B) Percentage of scn1a mutant larvae with epileptiform activity below (white area) or above the threshold value (black area). Single treatment with FA induced anti-epileptiform locomotor activity (FA), which was counteracted by combinatorial treatment with the 5-HT_1D-antagonist or the 5-HT_2C-antagonist or triple treatment of epileptiform compounds [σ₁-agonist and subtype selective 5-HT-antagonists (1D and 2C)]. A statistical difference is indicated by: ^∗p < 0.05 and ^∗∗∗p < 0.001 vs. VHC-treated controls (or FA-treated larvae). n = 16–30 ZF larvae for all experimental conditions.

FIGURE 5

Activity profile of FA [single treatment with FA and combinatorial treatment of FA with the sigma₁-agonist (σ₁) or subtype selective 5-HT-antagonists (1D, 2A, 2C, 2B) or triple treatment of epileptiform compounds [σ₁-agonist and subtype selective 5-HT-antagonists (1D and 2C)] (forebrain LFP recordings)]. Quantification of brain recordings of VHC-treated WT larvae [VHC(+/+), n = 42], VHC-treated scn1a mutant larvae [VHC(-/-), n = 41] and compound-treated scn1a mutant larvae [compound(-/-)]. (A) Frequency of epileptiform events during 10 minutes recording. (B) Mean cumulative duration of epileptiform events during 10 min recording. Single treatment with FA reduced epileptiform brain activity (n = 50), which was counteracted by combinatorial treatment with the σ₁-agonist (n = 13), the 5-HT_1D-antagonist (n = 18) or the 5-HT_2C-antagonist (n = 16). A statistical difference is indicated by ^∗p < 0.05 and ^∗∗∗p < 0.001. (C) Visualization of representative electrograms of 7 dpf ZF larvae: FA-treated scn1a mutant larva [FA(-/-)]; FA-treated scn1a mutant larva in combination with the σ₁-agonist [FA+ σ₁-agonist(-/-)] or triple treatment with epileptiform compounds [σ₁-agonist+5-HT_1D&2C-antagonists (-/-)]; scale bars: 1 mV, 30 s.

FIGURE 6

Activity profile of the sigma₁-agonist (σ₁) and subtype selective 5-HT-antagonists (1D, 2A, 2C, 2B) (locomotor behavioral assays). Locomotor activity was normalized against VHC-treated scn1a mutant larvae (-/-) and displayed as a percentage ± SD. (A1) Normalized locomotor activity of treated scn1a mutant larvae (-/-). (A2) Normalized locomotor activity of treated WT larvae (+/+). (B) Percentage of scn1a mutant larvae with epileptiform activity below (white area) or above the threshold value (black area). Single treatment with the σ₁-agonist (σ₁) and the subtype selective 5-HT-antagonists (1D, 2A, 2C, 2B) did not significantly affect the epileptiform locomotor behavior. A statistical difference is indicated by: ^∗p < 0.05 vs. VHC-treated controls. n = 20 ZF larvae for all experimental conditions.

FIGURE 7

Neurotransmitter content in head homogenates of ZF larvae (nmol/mg). Data for each condition were collected from eight samples with each six heads (n = 48). ^∗p < 0.05 and ^∗∗p < 0.01 indicate a statistically significant alteration of some neurotransmitters (Supplementary Table S6; Two-way ANOVA).