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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.

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Sorting of heterogeneous bursts into statistically different states. The left (A, B) and right (C, D) panels correspond to WT and Mutant data, respectively. Rows show the properties of the four different states (see Section Materials and Methods), as indicated. (A, C) Plots of the different average number of spikes per burst (SN), calculated for each state within four 2 h segments. Open and closed squares indicate, respectively, excitatory and inhibitory neurons. (B, D) Plot of the different time histograms of the average spikes/burst (bin was 0.1 s) corresponding to the indicated states. Open circles and continuous lines indicate, respectively, excitatory and inhibitory neurons. In the WT network we identified 69 unit (52 excitatory and 17 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 736, 755, 758 and 980. The average IBIs (inter burst intervals) were comprised between 8 and 10 s. In the Mutant network we identified 78 unit (63 excitatory and 15 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 422, 256, 299 and 282. The average IBIs were comprised between 22 and 26 s.
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Figure 2: Sorting of heterogeneous bursts into statistically different states. The left (A, B) and right (C, D) panels correspond to WT and Mutant data, respectively. Rows show the properties of the four different states (see Section Materials and Methods), as indicated. (A, C) Plots of the different average number of spikes per burst (SN), calculated for each state within four 2 h segments. Open and closed squares indicate, respectively, excitatory and inhibitory neurons. (B, D) Plot of the different time histograms of the average spikes/burst (bin was 0.1 s) corresponding to the indicated states. Open circles and continuous lines indicate, respectively, excitatory and inhibitory neurons. In the WT network we identified 69 unit (52 excitatory and 17 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 736, 755, 758 and 980. The average IBIs (inter burst intervals) were comprised between 8 and 10 s. In the Mutant network we identified 78 unit (63 excitatory and 15 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 422, 256, 299 and 282. The average IBIs were comprised between 22 and 26 s.

Mentions: Bursts were analyzed as previously described (Gullo et al., 2009, 2010). Bursts composed of more than two spikes were identified with Neuroexplorer. To the bursts containing exactly 2 spikes, we assigned a BD equal to their ISI and SN of 2. To single spikes, we assigned a BD of 2 ms and a SN of 1. The rationale for this procedure is as follows: (1) CNS neurons and particularly neocortical pyramidal neurons in vivo are tightly controlled by surrounding inhibition, and thus typically fire few spikes, and frequently single spikes (e.g., Pouille and Scanziani, 2001). A similar situation should be considered physiological in in vitro networks; (2) all units in which single spikes were occasionally observed were characterized by a majority of bursts containing two or more spikes, with an average SN always higher than 2; (3) the classical burst definition (SN ≥ 3) would lead to wrong estimates of SN; and (4) our networks were silent during the down-states. We discarded the units (1–2 in each network) that fired continuously. As is shown in the SN time histograms of Figures 2B,D only at the end of each burst the number of spikes becomes very small. On the contrary, the average SN values and their standard errors indicate that the cases of one or two spikes only are very rare.


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)

Sorting of heterogeneous bursts into statistically different states. The left (A, B) and right (C, D) panels correspond to WT and Mutant data, respectively. Rows show the properties of the four different states (see Section Materials and Methods), as indicated. (A, C) Plots of the different average number of spikes per burst (SN), calculated for each state within four 2 h segments. Open and closed squares indicate, respectively, excitatory and inhibitory neurons. (B, D) Plot of the different time histograms of the average spikes/burst (bin was 0.1 s) corresponding to the indicated states. Open circles and continuous lines indicate, respectively, excitatory and inhibitory neurons. In the WT network we identified 69 unit (52 excitatory and 17 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 736, 755, 758 and 980. The average IBIs (inter burst intervals) were comprised between 8 and 10 s. In the Mutant network we identified 78 unit (63 excitatory and 15 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 422, 256, 299 and 282. The average IBIs were comprised between 22 and 26 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Sorting of heterogeneous bursts into statistically different states. The left (A, B) and right (C, D) panels correspond to WT and Mutant data, respectively. Rows show the properties of the four different states (see Section Materials and Methods), as indicated. (A, C) Plots of the different average number of spikes per burst (SN), calculated for each state within four 2 h segments. Open and closed squares indicate, respectively, excitatory and inhibitory neurons. (B, D) Plot of the different time histograms of the average spikes/burst (bin was 0.1 s) corresponding to the indicated states. Open circles and continuous lines indicate, respectively, excitatory and inhibitory neurons. In the WT network we identified 69 unit (52 excitatory and 17 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 736, 755, 758 and 980. The average IBIs (inter burst intervals) were comprised between 8 and 10 s. In the Mutant network we identified 78 unit (63 excitatory and 15 inhibitory). The number of identified up-states in the four 2 h segments were, respectively, 422, 256, 299 and 282. The average IBIs were comprised between 22 and 26 s.
Mentions: Bursts were analyzed as previously described (Gullo et al., 2009, 2010). Bursts composed of more than two spikes were identified with Neuroexplorer. To the bursts containing exactly 2 spikes, we assigned a BD equal to their ISI and SN of 2. To single spikes, we assigned a BD of 2 ms and a SN of 1. The rationale for this procedure is as follows: (1) CNS neurons and particularly neocortical pyramidal neurons in vivo are tightly controlled by surrounding inhibition, and thus typically fire few spikes, and frequently single spikes (e.g., Pouille and Scanziani, 2001). A similar situation should be considered physiological in in vitro networks; (2) all units in which single spikes were occasionally observed were characterized by a majority of bursts containing two or more spikes, with an average SN always higher than 2; (3) the classical burst definition (SN ≥ 3) would lead to wrong estimates of SN; and (4) our networks were silent during the down-states. We discarded the units (1–2 in each network) that fired continuously. As is shown in the SN time histograms of Figures 2B,D only at the end of each burst the number of spikes becomes very small. On the contrary, the average SN values and their standard errors indicate that the cases of one or two spikes only are very rare.

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