Search

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.

The balance between glutamate- and GABA-mediated neurotransmission in the brain is fundamental in the nervous system, but it is regulated by the "tonic" release of a variety of endogenous factors. One such important group of molecules are the neurosteroids (NSs) which, similarly to benzodiazepines (BDZs), enhance GABAergic neurotransmission. The purpose of our work was to investigate, at in vivo physiologically relevant concentrations, the effects of NSs and BDZs as GABA modulators on dissociated neocortical neuron networks grown in long-term culture. We used a multi-electrode array (MEA) recording technique and a novel analysis that was able to both identify the action potentials of engaged excitatory and inhibitory neurons and to detect drug-induced network up-states (burst). We found that the NSs tetrahydrodeoxycorticosterone (THDOC) and allopregnanolone (ALLO) applied at low nanomolar concentrations, produced different modulatory effects on the two neuronal clusters. Conversely, at high concentrations (1 μM), both NSs, decreased excitatory and inhibitory neuron cluster excitability; however, even several hours after wash-out, the excitability of inhibitory neurons continued to be depressed, leading to a network long-term depression (LTD). The BDZs clonazepam (CLZ) and midazolam (MDZ) also decreased the network excitability, but only MDZ caused LTD of inhibitory neuron cluster. To investigate the origin of the LTD after MDZ application, we tested finasteride (FIN), an inhibitor of endogenous NSs synthesis. FIN did not prevent the LTD induced by MDZ, but surprisingly induced it after application of CLZ. The significance and possible mechanisms underlying these LTD effects of NSs and BDZs are discussed. Taken together, our results not only demonstrate that ex vivo networks show a sensitivity to NSs and BDZs comparable to that expressed in vivo, but also provide a new global in vitro description that can help in understanding their activity in more complex systems.

Distinguishing excitatory from inhibitory neurons with multielectrode array (MEA) recordings is a serious experimental challenge. The current methods, developed in vitro, mostly rely on spike waveform analysis. These however often display poor resolution and may produce errors caused by the variability of spike amplitudes and neuron shapes. Recent recordings in human brain suggest that the spike waveform features correlate with time-domain statistics such as spiking rate, autocorrelation, and coefficient of variation. However, no precise criteria are available to exactly assign identified units to specific neuronal types, either in vivo or in vitro. To solve this problem, we combined MEA recording with fluorescence imaging of neocortical cultures from mice expressing green fluorescent protein (GFP) in GABAergic cells. In this way, we could sort out "authentic excitatory neurons" (AENs) and "authentic inhibitory neurons" (AINs). We thus characterized 1275 units (from 405 electrodes, n = 10 experiments), based on autocorrelation, burst length, spike number (SN), spiking rate, squared coefficient of variation, and Fano factor (FF) (the ratio between spike-count variance and mean). These metrics differed by about one order of magnitude between AINs and AENs. In particular, the FF turned out to provide a firing code which exactly (no overlap) recognizes excitatory and inhibitory units. The difference in FF between all of the identified AEN and AIN groups was highly significant (p < 10(-8), ANOVA post-hoc Tukey test). Our results indicate a statistical metric-based approach to distinguish excitatory from inhibitory neurons independently from the spike width.

It has been demonstrated using single-cell and multiunit electrophysiology in layer III entorhinal cortex and disinhibited hippocampal CA3 slices that the balancing of the up-down activity is characterized by both GABA(A) and GABA(B) mechanisms. Here we report novel results obtained using multi-electrode array (60 electrodes) simultaneous recordings from reverberating postnatal neocortical networks containing 19.2 +/- 1.4% GABAergic neurons, typical of intact tissue. We observed that in each spontaneous active-state the total number of spikes in identified clusters of excitatory and inhibitory neurons is almost equal, thus suggesting a balanced average activity. Interestingly, in the active-state, the early phase is sustained by only 10% of the total spikes and the firing rate follows a sigmoidal regenerative mode up to peak at 35 ms with the number of excitatory spikes greater than inhibitory, therefore indicating an early unbalance. Concentration-response pharmacology of up- and down-state lifetimes in clusters of excitatory (n = 1067) and inhibitory (n = 305) cells suggests that, besides the GABA(A) and GABA(B) mechanisms, others such as GAT-1-mediated uptake, I(h), I(NaP) and I(M) ion channel activity, robustly govern both up- and down-activity. Some drugs resulted to affect up- and/or down-states with different IC(50)s, providing evidence that various mechanisms are involved. These results should reinforce not only the role of synchrony in CNS networks, but also the recognized analogies between the Hodgkin-Huxley action potential and the population bursts as basic mechanisms for originating membrane excitability and CNS network synchronization, respectively.