Reversal of pentylenetetrazole-altered swimming and neural activity-regulated gene expression in zebrafish larvae by valproic acid and valerian extract

Abstract

RationaleEthnopharmacology has documented hundreds of psychoactive plants awaiting exploitation for drug discovery. A robust and inexpensive in vivo system allowing systematic screening would be critical to exploiting this knowledge.ObjectiveThe objective of this study was to establish a cheap and accurate screening method which can be used for testing psychoactive efficacy of complex mixtures of unknown composition, like plant crude extracts.MethodsWe used automated recording of zebrafish larval swimming behavior during light vs. dark periods which we reproducibly altered with an anxiogenic compound, pentylenetetrazole (PTZ). First, we reversed this PTZ-altered swimming by co-treatment with a well-defined synthetic anxiolytic drug, valproic acid (VPA). Next, we aimed at reversing it by adding crude root extracts of Valeriana officinalis (Val) from which VPA was originally derived. Finally, we assessed how expression of neural activity-regulated genes (c-fos, npas4a, and bdnf) known to be upregulated by PTZ treatment was affected in the presence of Val.ResultsBoth VPA and Val significantly reversed the PTZ-altered swimming behaviors. Noticeably, Val at higher doses was affecting swimming independently of the presence of PTZ. A strong regulation of all three neural-activity genes was observed in Val-treated larvae which fully supported the behavioral results.ConclusionsWe demonstrated in a combined behavioral-molecular approach the strong psychoactivity of a natural extract of unknown composition made from V. officinalis. Our results highlight the efficacy and sensitivity of such an approach, therefore offering a novel in vivo screening system amenable to high-throughput testing of promising ethnobotanical candidates.Electronic supplementary materialThe online version of this article (doi:10.1007/s00213-016-4304-z) contains supplementary material, which is available to authorized users.

Hypo- or hyperactivity of zebrafish embryos provoked by neuroactive substances: a review on how experimental parameters impact the predictability of behavior changes

“…4). In the LMR-L/D, PTZ induces a behavior which is opposite to that observed in control embryos, i.e., high activity in dark period and low activity in light period for embryos in untreated solution [31,115]. Therefore, all hyperactivity effects were recorded during the light phase in the LMR-L/D tests with PTZ.…”

“…The text written on top of each bar represents the behavioral test method while the numbers represents the exposure duration (hpf ). References for each study can also be found in Additional file 2: Excel sheet 3 periods and PTZ has been reported to cause a reversal of the observed activity in control treatment, i.e., higher activity in dark and lower activity in light phase for nonexposed embryos [31,115]. Consequently, it is likely that PTZ induces a differentialresponse in light and dark phases and this effect is only observed under alternating light conditions.…”

Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2

“…Stock solutions were prepared in SW at 20 mM for VPA (Sigma #P4543) and 75 mM for PTZ (Sigma #P6500), 30 or in dimethyl sulfoxide at 10 mM for all CB2 ligands (Tocris bioscience: JWH-133 #1343, HU-308 #3088, AM-630 #1120, and SR-144528 #5039). Further dilutions were done in SW.…”

“…28 Homozygote larvae were viable without an overt phenotype and were raised into fertile and healthy adults over several generations, which were all completely lacking CB2. Next, we tested swimming behaviors in 6-day postfertilization (dpf) wild-type and homozygote larvae monitoring photo-dependent responses (PDR) 29,30 and measuring the center occupancy (CO) of wells providing an inverse measure of center avoidance. We found that CB2-KO were swimming significantly less in light and significantly more in dark periods with a decreased CO, when compared with wild-type larvae.…”

“…We found that CB2-KO were swimming significantly less in light and significantly more in dark periods with a decreased CO, when compared with wild-type larvae. When adding a broad-spectrum anxiolytic drug (valproic acid [VPA]) 30,31 just prior recording, we found that swimming activity and CO were strongly reduced in all animals in a similar manner, but swimming was slightly less and CO more diminished in mutants. When adding a classical anxiogenic drug, pentylenetetrazol (PTZ), a well-characterized GABA A inhibitor in many animal models, 32–36 we found that larvae lacking CB2 presented an increase in swimming activity and a decrease in CO when compared with wild type.…”