Limits...
Multi-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy.

Gullo F, Manfredi I, Lecchi M, Casari G, Wanke E, Becchetti A - Front Neural Circuits (2014)

Bottom Line: Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures.This opens the way to the study in vitro of the role of β2-V287L on synaptic formation.Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

ABSTRACT
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10-32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

Show MeSH

Related in: MedlinePlus

Effects of CBZ and GZ. (A) Superimposed cumulative histograms of the BD in the absence (control) and in the presence of 30 μM CBZ. Data refer to a typical experiment in which CBZ was applied for 8 h, after 8 h recording in control conditions. The inset shows the result of an exemplary experiment (1 out of 4) performed in WT networks (same symbols and scales as used for Mutant). (B) Superimposed cumulative histograms of the BD in the absence (2 h; control) and in the presence of 1 μM GZ (2 h). The networks recovered the initial level of activity, after washout (not shown). For both CBZ and GZ, data are representative of 3 experiments carried out on mice from different litters. In both A and B the application of the non-parametric Kruskal-Wallis tests resulted in p-values < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109561&req=5

Figure 5: Effects of CBZ and GZ. (A) Superimposed cumulative histograms of the BD in the absence (control) and in the presence of 30 μM CBZ. Data refer to a typical experiment in which CBZ was applied for 8 h, after 8 h recording in control conditions. The inset shows the result of an exemplary experiment (1 out of 4) performed in WT networks (same symbols and scales as used for Mutant). (B) Superimposed cumulative histograms of the BD in the absence (2 h; control) and in the presence of 1 μM GZ (2 h). The networks recovered the initial level of activity, after washout (not shown). For both CBZ and GZ, data are representative of 3 experiments carried out on mice from different litters. In both A and B the application of the non-parametric Kruskal-Wallis tests resulted in p-values < 0.0001.

Mentions: Data are given as mean values ± standard error of the mean, with n indicating the number of experiments. Statistical significance for normally distributed data was assessed with OriginPro 8.0 (OriginLab Co., Northampthon, MA), by using ANOVA test with the Bonferroni post-hocp-values. Normality was tested by the Kolmogorov-Smirnov test. Non-normally distributed data were analyzed with non parametric tests, by using the XLSTAT-Pro software (Addinsoft, USA). In Figure 2 we applied the Mann-Whitney, the Wilcoxon Signed Rank and Kruskal-Wallis tests, which gave similar results. The BD histograms from different experiments (Figures 3–5) generally did not present an identical number of data points (bins). Therefore, to apply the Wilcoxon-Sign test, the bin number was uniformed by adding zeros in empty bins. Instead, to directly compare the real BDs over many time segments, we used the Kruskal-Wallis test (the non-parametric equivalent of ANOVA). The data heterogeneity (e.g., Figure 4) was further characterized by multiple comparison methods such as the Dunn’s and the Steel-Dwass-Critchlow-Finger’s (with Bonferroni correction; Hollander and Wolfe, 1999).


Multi-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy.

Gullo F, Manfredi I, Lecchi M, Casari G, Wanke E, Becchetti A - Front Neural Circuits (2014)

Effects of CBZ and GZ. (A) Superimposed cumulative histograms of the BD in the absence (control) and in the presence of 30 μM CBZ. Data refer to a typical experiment in which CBZ was applied for 8 h, after 8 h recording in control conditions. The inset shows the result of an exemplary experiment (1 out of 4) performed in WT networks (same symbols and scales as used for Mutant). (B) Superimposed cumulative histograms of the BD in the absence (2 h; control) and in the presence of 1 μM GZ (2 h). The networks recovered the initial level of activity, after washout (not shown). For both CBZ and GZ, data are representative of 3 experiments carried out on mice from different litters. In both A and B the application of the non-parametric Kruskal-Wallis tests resulted in p-values < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109561&req=5

Figure 5: Effects of CBZ and GZ. (A) Superimposed cumulative histograms of the BD in the absence (control) and in the presence of 30 μM CBZ. Data refer to a typical experiment in which CBZ was applied for 8 h, after 8 h recording in control conditions. The inset shows the result of an exemplary experiment (1 out of 4) performed in WT networks (same symbols and scales as used for Mutant). (B) Superimposed cumulative histograms of the BD in the absence (2 h; control) and in the presence of 1 μM GZ (2 h). The networks recovered the initial level of activity, after washout (not shown). For both CBZ and GZ, data are representative of 3 experiments carried out on mice from different litters. In both A and B the application of the non-parametric Kruskal-Wallis tests resulted in p-values < 0.0001.
Mentions: Data are given as mean values ± standard error of the mean, with n indicating the number of experiments. Statistical significance for normally distributed data was assessed with OriginPro 8.0 (OriginLab Co., Northampthon, MA), by using ANOVA test with the Bonferroni post-hocp-values. Normality was tested by the Kolmogorov-Smirnov test. Non-normally distributed data were analyzed with non parametric tests, by using the XLSTAT-Pro software (Addinsoft, USA). In Figure 2 we applied the Mann-Whitney, the Wilcoxon Signed Rank and Kruskal-Wallis tests, which gave similar results. The BD histograms from different experiments (Figures 3–5) generally did not present an identical number of data points (bins). Therefore, to apply the Wilcoxon-Sign test, the bin number was uniformed by adding zeros in empty bins. Instead, to directly compare the real BDs over many time segments, we used the Kruskal-Wallis test (the non-parametric equivalent of ANOVA). The data heterogeneity (e.g., Figure 4) was further characterized by multiple comparison methods such as the Dunn’s and the Steel-Dwass-Critchlow-Finger’s (with Bonferroni correction; Hollander and Wolfe, 1999).

Bottom Line: Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures.This opens the way to the study in vitro of the role of β2-V287L on synaptic formation.Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

ABSTRACT
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10-32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

Show MeSH
Related in: MedlinePlus