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

Elementary properties of the short and long up-states. (A) Each column represents an up-state (called network burst or global burst) and each row (here highlighted by a horizontal line) shows the trace recorded by one electrode. The panel shows a raster plot representing the multi-electrode activity recorded during 100 s. Each small vertical tick in a row is the timestamp representing a single spike. If the same electrode acquires spikes from more than one identified unit (neuron), the ticks corresponding to different units are indicated by different colors. Notice the very long burst in the middle of the raster plot. The spike waveforms recorded from electrode 21 are shown in the 1st, 2nd, 3rd and 4th bottom inset, at increasing time scale magnifications, as indicated. The last inset on the bottom shows 40 ms continuous recording. (B–C) Plots of the spike waveforms on a time-scale of 1.2 ms. The spikes shown in the 4th inset are superimposed in panel B and originated from the very long up-state. The spikes of all the very short network bursts following the long one are superimposed in panel C. Notice the similarity between all of these waveforms. (D–E) Raster plots representative of other experiments in Mutant cultures (duration and timescale were as in A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Elementary properties of the short and long up-states. (A) Each column represents an up-state (called network burst or global burst) and each row (here highlighted by a horizontal line) shows the trace recorded by one electrode. The panel shows a raster plot representing the multi-electrode activity recorded during 100 s. Each small vertical tick in a row is the timestamp representing a single spike. If the same electrode acquires spikes from more than one identified unit (neuron), the ticks corresponding to different units are indicated by different colors. Notice the very long burst in the middle of the raster plot. The spike waveforms recorded from electrode 21 are shown in the 1st, 2nd, 3rd and 4th bottom inset, at increasing time scale magnifications, as indicated. The last inset on the bottom shows 40 ms continuous recording. (B–C) Plots of the spike waveforms on a time-scale of 1.2 ms. The spikes shown in the 4th inset are superimposed in panel B and originated from the very long up-state. The spikes of all the very short network bursts following the long one are superimposed in panel C. Notice the similarity between all of these waveforms. (D–E) Raster plots representative of other experiments in Mutant cultures (duration and timescale were as in A).

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)

Elementary properties of the short and long up-states. (A) Each column represents an up-state (called network burst or global burst) and each row (here highlighted by a horizontal line) shows the trace recorded by one electrode. The panel shows a raster plot representing the multi-electrode activity recorded during 100 s. Each small vertical tick in a row is the timestamp representing a single spike. If the same electrode acquires spikes from more than one identified unit (neuron), the ticks corresponding to different units are indicated by different colors. Notice the very long burst in the middle of the raster plot. The spike waveforms recorded from electrode 21 are shown in the 1st, 2nd, 3rd and 4th bottom inset, at increasing time scale magnifications, as indicated. The last inset on the bottom shows 40 ms continuous recording. (B–C) Plots of the spike waveforms on a time-scale of 1.2 ms. The spikes shown in the 4th inset are superimposed in panel B and originated from the very long up-state. The spikes of all the very short network bursts following the long one are superimposed in panel C. Notice the similarity between all of these waveforms. (D–E) Raster plots representative of other experiments in Mutant cultures (duration and timescale were as in A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Elementary properties of the short and long up-states. (A) Each column represents an up-state (called network burst or global burst) and each row (here highlighted by a horizontal line) shows the trace recorded by one electrode. The panel shows a raster plot representing the multi-electrode activity recorded during 100 s. Each small vertical tick in a row is the timestamp representing a single spike. If the same electrode acquires spikes from more than one identified unit (neuron), the ticks corresponding to different units are indicated by different colors. Notice the very long burst in the middle of the raster plot. The spike waveforms recorded from electrode 21 are shown in the 1st, 2nd, 3rd and 4th bottom inset, at increasing time scale magnifications, as indicated. The last inset on the bottom shows 40 ms continuous recording. (B–C) Plots of the spike waveforms on a time-scale of 1.2 ms. The spikes shown in the 4th inset are superimposed in panel B and originated from the very long up-state. The spikes of all the very short network bursts following the long one are superimposed in panel C. Notice the similarity between all of these waveforms. (D–E) Raster plots representative of other experiments in Mutant cultures (duration and timescale were as in A).
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