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. 2020 Mar;19(3):2326-2334.
doi: 10.3892/etm.2020.8476. Epub 2020 Jan 28.

Effects of tenuifolin on rest/wake behaviour in zebrafish

Zi-Wen Chen  1 , Chao-Bao Peng  2 , Zhong Pei  3 , Meng-Ruo Zhang  1 , Tian-Chan Yun  1 , Zhi-Min Yang  4 , Fu-Ping Xu  4
Affiliations

Effects of tenuifolin on rest/wake behaviour in zebrafish

Zi-Wen Chen et al. Exp Ther Med. 2020 Mar.

Abstract

Insomnia is a common sleep disorder with a high prevalence and substantial adverse consequences. There is growing interest in identifying novel therapeutics from herbal medicine. Tenuifolin is a major constituent of the well-known anti-insomnia herb Radix Polygala. The present study investigated the neural activity in response to tenuifolin during rest/wake behaviour in zebrafish and identified the potential biological signalling pathways involved. An automatic video tracking system was used to monitor the behavioural response of zebrafish larvae for 24 h after treatment with tenuifolin. In total, six rest/wake parameters were measured and visualized with a behavioural fingerprint. Time series analysis was conducted by averaging the total rest and waking activity in 10 min intervals. A correlation analysis was performed between tenuifolin and well-known compounds to analyse the underlying biological signalling pathways. Reverse transcription-quantitative PCR was also performed to detect the effects of tenuifolin on the transcription of interesting genes associated with the signalling pathways that were potentially involved. The present results suggested tenuifolin significantly increased the total rest time during the dark phase, with a slight effect on the waking activity in zebrafish larvae. This behavioural phenotype induced by tenuifolin is similar to that of selective serotonin reuptake inhibitors and gamma-aminobutyric acid (GABA) agonists. Furthermore, the expression levels of GABA transporter 1 were significantly increased after tenuifolin treatment. No significant difference was determined in other associated genes in untreated control and tenuifolin-treated larvae. The present results suggested that tenuifolin caused sleep-promoting activity in zebrafish and that these effects may be mediated by the serotoninergic systems and the GABAergic systems.

Keywords: behavioural profiles; herbal medicine; neural signalling pathways; sleep; tenuifolin; zebrafish.

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Figures

Figure 1.
Figure 1.
Chemical structure of tenuifolin, C36H56O12.
Figure 2.
Figure 2.
Behavioural fingerprint induced by tenuifolin in zebrafish larvae. Each row represents a treatment and each column represents a behavioural parameter. Each measurement was normalized as standard deviations from the corresponding untreated control and is represented as different colours: Red, > untreated controls; black, untreated controls; yellow, < untreated controls.
Figure 3.
Figure 3.
Time series analysis of total rest and waking activity. The average rest and waking activity in 10 min intervals were calculated and normalized to the matched untreated control. (A) The normalized total rest and waking activity of larvae treated with 1 µM tenuifolin. The red trace indicates the average of the 1 µM tenuifolin group and the black trace indicates the average of the untreated control group. (B) The normalized total rest and waking activity of larvae treated with 10 µM tenuifolin. The red trace indicates the average of the 10 µM tenuifolin group and the black trace indicates the average of the untreated control group. (C) The normalized total rest and waking activity of larvae treated with 20 µM tenuifolin. The red trace indicates the average of the 20 µM tenuifolin group and the black trace indicates the average of the untreated control group. (D) The normalized total rest and waking activity of larvae treated with 30 µM tenuifolin. The red trace indicates the average of the 30 µM tenuifolin group and the black trace indicates the average of the untreated control group. (E) The normalized total rest and waking activity of larvae treated with melatonin. The red trace indicates the average of the melatonin group and the black trace indicates the average of the untreated control group.
Figure 4.
Figure 4.
(A-B) Quantitative analysis of total rest and waking activity. Data are presented as mean ± SEM. *P<0.05 vs. the untreated control group. (A) Quantitative analysis of total rest. (B) Quantitative analysis of waking activity.
Figure 5.
Figure 5.
Correlation analysis of tenuifolin with known compounds. Each row represents a different compound and its corresponding target, belonging to a different signalling pathway. Each column indicates a different tenuifolin concentration. Rectangles in different colours represent the correlation coefficient of a pair of drugs: Red, high correlation; blue, low correlation. The results with a Pearson correlation coefficient >0.5 are presented.
Figure 6.
Figure 6.
Effects on total rest after cotreatment with tenuifolin and picrotoxin. Data are presented as mean ± SEM. *P<0.05 vs. the untreated control group. #P<0.05 vs. 30 µM tenuifolin.
Figure 7.
Figure 7.
Expression of genes associated with the serotoninergic and GABAergic signalling pathways after tenuifolin treatment. The transcript levels of different genes were normalized to that of β-act. (A) Expression of genes associated with the serotoninergic signalling pathways after tenuifolin treatment. (B) Expression of genes associated with the GABAergic signalling pathways after tenuifolin treatment. Data are presented as mean ± SEM. *P<0.05 vs. the untreated control group. β-act, β-actin; GABA, gamma-aminobutyric acid; sert, serotonin transporter; 5-HT1A serotonin 1A; gad1, glutamate decarboxylase 1; gabra1, GABAA receptor α1; gat1, GABA transporter 1.
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