Multisite Field Potential Recordings Research Articles

G005 In vitro simulation of spiral waves in cardiomyocyte networks using multi-electrode array technology

Atrial and ventricular fibrillations are thought to be caused by multiple chaotically wandering wavelets of excitation of unknown origin. We aimed thus to provide new insights into the cellular origin of the fibrillation phenomenon by exploring the impulse propagation between cardiac myocytes in confluent monolayers of cultured cardiomyocytes (CM), which beat spontaneously and synchronously. Multisite field potentials have been recorded using microelectrode arrays (MEA) technology in basal conditions and in proarrhythmic conditions. CM were grown on multi-electrode arrays (MEA) allowing non-invasive synchronous multifocal field potential (FP) recordings. The MEA consists of 60 substrate-integrated microelectrode arrays (8 × 8 matrix, 30 μm electrode diameter, 200μm inter-electrode distance). Data were real time acquired and analyzed with a customized platform programmed with MATLAB (Mathworks) in order to provide two-dimensional electrophysiological maps derived from these multisite FP recordings, in particular the contour of the FP propagation wavefront during each period. The sets of activation maps were then used to reconstruct videos revealing multicycle spatiotemporal patterns. In basal condition, the observation of these activation maps indicated that the spontaneous FP spikes propagated following linear path, with very stable periodic characteristics. Cardiomyocytes were then stimulated by an external electrical signal, consisting in a stimulation train (burst of 200 mV at 100 Hz during 5 min) in one point at the edge of the MEA. After this stimulation protocol, the recorded electrical activities became irregular, as confirmed by Poincaré maps of the FP periods. Moreover, spiral waves (SW) appeared in the cardiac cell network. These SW were unstable in location and could move inside or outside the recording area. SW had a mean radius of 400×100 μm and a mean angular velocity of 225×30 rotations per minute. Unstable reentrant and colliding wavefronts were also observed. To conclude, SW can be precisely characterized using a MEA data acquisition system. Therefore, within the limitations inherent to the preparation used, the cultured CM monolayer is a controlled experimental model that may be useful for further studies on the basic aspects of fibrillation and defibrillation.

Excitation spread in cardiac myocyte cultures using paired microelectrode and microelectrode array recordings

Abnormal cell–cell coupling is associated with increased arrhythmic risk, but the detailed electrophysiological understanding of this arrhythmic propensity is still unclear. Therefore, we performed a multiparametric study of excitation spread with cellular resolution in confluent monolayers of cultured cardiomyocytes (CM). Using simultaneous intracellular recordings of action potentials in individual CM pairs, the mean conduction velocity was measured at 30.4±2.2 cm/s (mean±SD). Moreover, in spite of a stable interbeat period and action potential (AP) time parameters, we observed slight periodic spontaneous advances/delays in the interspike time lag, suggesting natural fluctuations in cell coupling and/or in conduction pathway. To check this, synchronous multifocal field potential (FP) recordings were done from CM monolayers grown on substrate-integrated microelectrode arrays (MEA). These multisite FP recordings confirmed that AP parameters (rate, duration, voltage change velocity) were homogenous throughout the recording area. Conversely, the sequential mappings of the delays of activation calculated from the multielectrode matrix revealed for the first time random and iterative cycle-to-cycle changes in the direction of excitation spread. These spontaneous variations in the cardiac impulse propagation pathways may be a safety process protecting from microscopically discontinuous conduction, and abnormality of this natural process could contribute to the genesis of some heart arrhythmias.

Early developmental alterations of low-Mg 2+-induced epileptiform activity in the intact corticohippocampal formation of the newborn mouse in vitro

The generation, propagation and pharmacological properties of low-Mg 2+-induced epileptiform activity were examined in the intact corticohippocampal formation (CHF) of the newborn (P0–4) mouse in vitro. Multi-site field potential recordings in dentate gyrus (DG), CA3, CA1, entorhinal cortex (EC) and temporal cortex (TC) revealed in 0.2 mM Mg 2+-containing ACSF a stable pattern of spontaneous epileptiform activity consisting of recurrent ictal-like events (ILEs) and interictal events (IEs). Although this activity could be consistently observed as early as P0, ILEs were smaller in amplitude, less frequent and showed a slower onset in P0–2 as compared to P3–4 animals. In all age groups, epileptiform events were largest in CA3 and smallest in EC and TC. A specific pacemaker region could not be identified since ILEs appeared simultaneously at all recording sites. Reducing the extracellular Mg 2+ concentration to 0.1 mM or nominally zero caused an increase in ILE frequency. Pharmacological studies in the P3–4 age group with 0.2 mM Mg 2+ revealed a complete blockade of the ILEs by an NMDA receptor antagonist and a pronounced suppression of epileptiform activity by an AMPA/kainate antagonist. Application of a GABA-A receptor antagonist induced repetitive bursts of interictal discharges, which persisted for at least 1.5 h after washout of the antagonist. Our data demonstrate that the intact CHF in vitro preparation of the newborn mouse offers a most valuable model to study epileptiform activity in the immature limbic system.

Two reentrant pathways in the hippocampal-entorhinal system.

The entorhinal cortex has long been recognized as an important interface between the hippocampal formation and the neocortex. The notion of bidirectional connections between the entorhinal cortex and the hippocampal formation have led to the suggestion that hippocampal output originating in CA1 and subiculum may reenter hippocampal subfields via the entorhinal cortex. To investigate this, we used simultaneous multi-site field potential recordings and current source density analysis in the entorhinal cortex and hippocampal formation of the rat in vivo. Under ketamine/xylazine anesthesia, we found that repetitive stimulation of subiculum or Schaffer collaterals facilitated entorhinal responses, such that a population spike appeared in layer III. In addition, a current sink in stratum lacunosum-moleculare of area CA1 was found, that followed responses in the entorhinal cortex, indicating reentrance into this area. Responses indicating reentrance in the dentate gyrus were not found under ketamine/xylazine anesthesia, but were readily evoked under urethane anesthesia. Reentrance into CA1 was also encountered under urethane anesthesia. These results suggest that parallel, but possibly functionally distinct, connections are present between the output of the hippocampal formation and cells in layers III and II of the entorhinal cortex that project to area CA1 and the dentate gyrus, respectively.

Neuronal and Glial Membrane Potentials during Sleep and Paroxysmal Oscillations in the Neocortex

This study investigated the fluctuations in the membrane potential of cortical neurons and glial cells during the slow sleep oscillation and spike-wave (SW) seizures. We performed dual neuron-glia intracellular recordings together with multisite field potential recordings from cortical suprasylvian association areas 5 and 7 of cats under ketamine-xylazine anesthesia. Electrical stimuli applied to the cortex elicited responses consisting of a biphasic depolarization in glial cells, which was associated with an EPSP-IPSP sequence in neurons. During the slow (<1 Hz) oscillation, extracellular measurements of the potassium concentration revealed periodic increases with an amplitude of 1-2 mm, similar in shape to glial activities. We suggest that, through their uptake mechanisms, glia cells modulate the neuronal excitability and contribute to the pacing of the slow oscillation. The slow oscillation often evolved into SW paroxysms, mimicking sleep-triggered seizures. This transition was associated with increased coupling between the depolarizing events in neurons and glial cells. During seizures, the glial membrane potential displayed phasic negative events related to the onset of the paroxysmal depolarizing shifts in neurons. These events were not voltage dependent and increased their incidence and amplitude with the development of the seizure. It is suggested that the intraglial transient negativities represent field reflections of synchronized neuronal potentials. We propose that the mechanisms underlying the neuron-glia communication include, besides the traditional neurotransmitter- and ion-mediated pathways, field effects crossing their membranes as a function of the state of the cortical network.

Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.

We examined the generation, propagation and pharmacology of 4-aminopyridine (4-AP)-induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0-7) rat in vitro. Whole-cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4-AP-induced EA as early as P0-1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3-4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo-temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4-AP-induced EA decreased with age from 21.2 +/- 1.6 min at P0-1 to 4.7 +/- 0.63 min at P6-7, the seizure duration increased in the same age groups from 98 +/- 14 s to 269.4 +/- 85.9 s, respectively. The EA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by DL-2-amino-5-phosphonovaleric acid (APV), (+)-MK-801 hydrogen maleate (MK-801) or (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), suggesting that they were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor-mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.