Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Epilepsy, a clinical diagnosis characterised by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies since X. laevis is a well-characterised and genetically tractable model organism.
FIGURE 1. Pipeline for behavioural analysis of tadpoles induced to acute seizure activity with PTZ or 4‐AP. Xenopus laevis wild type embryos were raised to stage 47, at which point they will progress no further without feeding. Each tadpole was placed in its own well in a 24‐well plate and behaviour recorded for 30 min. Videos were analysed offline using TopScan software.
FIGURE 2. PTZ and 4‐AP induce different patterns of swimming behaviour. (a) Typical behaviours of PTZ‐ and 4‐AP‐treated tadpoles. Multiple consecutive frames are overlayed for UTB, Circling and Darting, with arrows indicating the sequence of frames. (b) Ethograms created by manual analysis of 10 tadpoles treated with either 5 mM PTZ or 0.5 mM 4‐AP to induce acute seizures. Circle diameter represents the mean frequency of each behaviour (see Table 1). Transitions between different behaviours are represented by arrows. Thicker arrows and closer circles indicates transitions between the two behaviours are more frequent. Data can be found in Table 1 and the Supplemental file 2.
FIGURE 3. The anti‐seizure drug VPA reduces 4‐AP‐induced C‐shaped contractions (CSC) and 4‐AP‐ and PTZ‐induced darting behaviour. Scatterplots showing manually counted CSC, mean velocity of tadpoles (cm/s), percentage of time spent circling and percentage of time spent darting. Black line indicates the mean and error bars are SEM. (a) Tadpoles treated with 5 mM PTZ with or without 5 mM VPA, N = 10 for each group, compared using unpaired t‐test (CSC, circling, darting) or Mann–Whitney test (velocity). (b) Tadpoles treated with 4‐AP, with or without VPA at two concentrations, 5 and 10 mM. Statistical analysis was by 1‐way ANOVA with Tukey's post hoc test of all means (circling) and by Kruskal–Wallis with Dunn's post hoc test (CSC, darting, velocity). N = 10 tadpoles in each group. Statistically different groups are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001. Data can be found in the Supplemental file 2.
FIGURE 4. Automatic analysis shows that VPA reduces PTZ‐ and 4‐AP‐induced swimming behaviours. (a) Representative examples of individual tadpole movement trajectories each treatment group, over the 30 min of the trial (treatment is colour‐coded as in b). (b) Box plots of average tadpole swimming velocity (cm/s), percentage of time spent swimming in rapid circles and percentage of time spent darting. Middle line indicates the median, upper and lower lines indicate the upper and lower quartile, and the whiskers indicate the minimum and maximum values. Kruskal–Wallis test with Dunn's post hoc testing of all means was used to analyse each dataset. Groups that share a lower case letter are not significantly different from one another whereas groups which are significantly different are indicated by a different lower case letter. N = number of tadpoles, VPA was used at 5 or 10 mM, PTZ at 5 mM and 4‐AP at 0.5 mM. Raw data can be found in Supplemental file 2.
FIGURE 5. Time to first 4‐AP induced circling or darting event is longer in the presence of anti‐seizure drug VPA. Kaplan–Meier plots where time to first seizure event (circling or darting) has been substituted for death. Tadpoles were censored if they did not have any events in the 30 min timespan and curves were compared with log‐rank (Mantel‐Cox) test. (a) Time to first circling event was longer for PTZ tadpoles co‐treated with VPA. (χ
2 = 4.5, df = 1, p = 0.033). (b) Tadpoles induced with 4‐AP all had at least one circling event, but VPA increased latency to first event, and this effect increased with dose (χ
2 = 63.3, df = 2, p < 0.0001). (c) The time to first darting effect induced by PTZ was not altered by VPA (χ
2 = 0.1, df = 1, p = 0.740). (d) Time to first darting event induced by 4‐AP was significantly longer in the presence of VPA (χ
2 = 129.2, df = 2, p < 0.0001) Group sizes: PTZ N = 84, PTZ + VPA N = 48, 4‐AP N = 124, 4‐AP + 5 mM VPA N = 124 4‐AP + 10 mM VPA, N = 96. Raw data can be found in Supplemental file 2. *p < 0.05, ****p < 0.0001.
