4 research outputs found
Exact distinction of excitatory and inhibitory neurons in neural networks: a study with GAD67-GFP neurons optically and electrophysiologically recognized on multi-electrode arrays
No full textDistinguishing 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, spiking rate, squared coefficient of variation and Fano factor (the ratio between spike-count variance and mean). These metrics differed by about one order of magnitude between AINs and AENs. In particular, the Fano factor turned out to provide a firing code which exactly (no overlap) recognizes excitatory and inhibitory units. The difference in Fano factor 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
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.
No full textAutosomal 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, thereby reducing the number of in vivo experiments
Atypical seizure-like activity in cortical reverberating networks in-vitro can be caused by LPS-induced inflammation: a multi-electrode array study from a hundred neurons
No full textWe show here that a mild sterile inflammation induced by the endotoxin lipopolysaccharide (LPS), in a neuron/astrocyte/microglial cortical network, modulates neuronal excitability and can initiate long-duration burst events resembling epileptiform seizures, a recognized feature of various central nervous neurodegenerative, neurological and acute systemic diseases associated with neuroinflammation. To study this action, we simultaneously analyzed the reverberating bursting activity of a hundred neurons by using in vitro multi-electrode array (MEA) methods. ~5 hours after LPS application, we observed a net increase in the average number of spikes elicited in engaged cells and within each burst, but no changes neither in spike waveforms nor in burst rate. This effect was characterized by a slow, two-fold exponential increase of the burst duration and the appearance of rarely occurring long-burst events that were never seen during control recordings. These changes and the time-course of microglia-released proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), were blocked by pre-treatment with 50 nM minocycline, an established anti-inflammatory agent which was inactive when applied alone. Assay experiments also revealed that application of 60 pM exogenous TNF-α after 12-15 h, produced non-washable changes of neuronal excitability, completely different from those induced by LPS, suggesting that TNF-α release alone was not responsible for our observed findings. Our results indicate that the link between neuroinflammation and hyperexcitability can be unveiled by studying the long-term activity of in vitro neuronal/astrocyte/microglial networks
Novel modulatory effects of neurosteroids and benzodiazepines on excitatory and inhibitory neurons excitability: a multi-electrode array (MEA) recording study"
No full textThe 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 nM 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 washout, 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
