Extracellularly Recorded Action Potentials Research Articles

Rapid Prototyping of Ultralow‐Cost, Inkjet‐Printed Carbon Microelectrodes for Flexible Bioelectronic Devices

AbstractGaining better understanding of the human brain using chip‐based devices and promoting the recovery of lost biological functionality through implants are long pursued endeavors driven by advances in material science, bioelectronics, and the advancing silicon technology. While conventional bioelectronic and neuroelectronic devices typically rely on cleanroom‐based processing, a rapid prototyping technique is proposed that is based on high‐resolution inkjet printing featuring nanoporous carbon electrodes that yield excellent cell–chip coupling. This study aims to overcome two major limitations of conventional approaches that make the development of neuroelectronic devices very challenging and limit a wider use within the research community as well as industry: high costs and lack of rapid prototyping capabilities. These challenges are addressed with an all‐printed, high‐resolution approach that makes use of flexible polymer substrates and is fabricated on a fully digital printing platform. The manufacturing of a chip consumes less than 60 min and costs a few cents per chip. This study introduces nanoporous carbon as a cell‐interfacing electrode material that features outstanding properties for extracellular recording of action potentials and stimulation indicating that the printed carbon chips have the means to be used as a versatile neuroelectronic tool for in vitro and in vivo studies.

Neurospheres on Patterned PEDOT:PSS Microelectrode Arrays Enhance Electrophysiology Recordings

AbstractMicroelectrode arrays (MEAs) are a versatile diagnostic tool to study neural networks. Culture of primary neurons on these platforms allows for extracellular recordings of action potentials. Despite many advances made in the technology to improve such recordings, the recording yield on MEAs remains sparse. Here, enhanced recording yield is shown induced by varying cell densities on poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate)‐coated MEAs. It is demonstrated that high cell densities (900 cells mm−2) of primary cortical cells increase the number of recording electrodes by 53.1% ± 11.3%, compared with low cell densities (500 cells mm−2) with 6.3% ± 1.4%. To further improve performance, 3D clusters known as neurospheres are cultured on the MEAs, significantly increasing single unit activity recordings. Extensive spike sorting is performed to analyze the unit activity recording multiple neurons with a single microelectrode. Finally, patterning of polyethylene glycol diacrylate through laser ablation is demonstrated, as a means to more precisely confine neurospheres on top of the electrodes. The possibility of recording single neurons with multiple neighboring electrodes is shown. Overall, a total recording yield of 21.4% is achieved, with more than 90% obtained from electrodes with neurospheres, maximizing the functionality of these planar MEAs as effective tools to study pharmacology‐based effects on neural networks.

All-inkjet-printed gold microelectrode arrays for extracellular recording of action potentials

Inkjet printing is an attractive method for cost-effective additive manufacturing of electronic devices. Especially for applications where disposable sensor systems are of interest, it is a promising tool since it enables the production of low-cost and flexible devices. In this work, we report the fabrication of a disposable microelectrode array (MEA) using solely inkjet printing technology. The MEAs were fabricated with two different functional inks, a self-made gold ink to print conductive feedlines and electrodes and a polymer-based ink to add a dielectric layer for insulation of the feedlines. We printed different MEA designs of up to 64 electrodes with a minimum lateral spacing of 200 μm and a minimum electrode diameter of ∼31 μm. As a proof-of-concept, extracellular recordings of action potentials from cardiomyocyte-like HL-1 cells were performed using the all-printed devices. Furthermore, we stimulated the cells during the recordings with noradrenaline, which led to an increase in the recorded beating frequency of the cells. The results demonstrate the feasibility of inkjet printing gold MEAs for cell-based bioelectronics.

The Transition from Stochastic to Collective Activation of Nicotinic Acetylcholine Receptors Facilitates Information Transmission Within in vitro Hippocampal Networks

Although modulatory in nature, nicotinic acetylcholine receptors (nAChRs) play a major role in the central nervous system, making them one of the most studied receptor class. While both activation and desensitization are phenomena associated with the functions of individual receptors, their effects will clearly manifest on the network/circuit spatial scale. In this study, nicotine was used to investigate the impact of activation and desensitization of nAChRs on cultured hippocampal networks. We assess network dynamics via the recording of extracellular action potentials using a microelectrode array system (MEA). We bath applied either a single dose or two successive doses of increasing concentration of nicotine. We hypothesize that a high concentration of nicotine will facilitate information transmission but pre‐incubation of the network with a low concentration of nicotine would attenuate facilitation. We show that the low concentration has minimal effects on network activity and we call this a network‐desensitized state. A high concentration significantly impacted both spiking and bursting activity and we call this a network‐activated state. Interestingly, a low concentration pre‐incubation of nicotine blocked or significantly diminished activity when followed by a high concentration. Lastly, network synchronization as well as network efficiency increased with the large nicotine concentration. This suggests that although fleeting, the process of global receptor activation has a longer lasting impact on the network than a stochastic receptor activation.Support or Funding InformationNational Science Foundation grant no. PHY‐1205919

Expression of the K+/Cl− cotransporter, KCC2, in cerebellar Purkinje cells is regulated by group-I metabotropic glutamate receptors

The neuronal K+/Cl− symporter, KCC2, shapes synaptic responses mediated by Cl−-permeant GABAA receptors. Moving from the evidence that excitatory neurotransmission drives changes in KCC2 expression in cerebellar neurons, we studied the regulation of KCC2 expression by group-I metabotropic glutamate (mGlu) receptors in the cerebellum of adult mice. Mice lacking mGlu5 receptors showed a large reduction in cerebellar KCC2 protein levels and a loss of KCC2 immunoreactivity in Purkinje cells. Similar changes were seen in mice treated with the mGlu5 receptor antagonist, MPEP, whereas treatment with the mGlu5 receptor positive allosteric modulator (PAM), VU0360172, increased KCC2 expression. In contrast, pharmacological inhibition of mGlu1 receptors with JNJ16259685 enhanced cerebellar KCC2 protein levels and KCC2 immunoreactivity in Purkinje cells, whereas treatment with the mGlu1 receptor PAM, RO0711401, reduced KCC2 expression. To examine whether the reduction in KCC2 expression caused by the absence or the inhibition of mGlu5 receptors could affect GABAergic transmission, we performed electrophysiological and behavioral studies. Recording of extracellular action potentials in Purkinje cells showed that the inhibitory effect of the GABAA receptor agonist, muscimol, was lost in cerebellar slices prepared from mGlu5−/− mice or from mice treated systemically with MPEP, in line with the reduction in KCC2 expression. Similarly, motor impairment caused by the GABAA receptor PAM, diazepam, was attenuated in mice pre-treated with MPEP. These findings disclose a novel function of mGlu5 receptors in the cerebellum and suggest that mGlu5 receptor ligands might influence GABAergic transmission in the cerebellum and affect motor responses to GABA-mimetic drugs.This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’.

Unsupervised neural spike sorting for high-density microelectrode arrays with convolutive independent component analysis

BackgroundUnsupervised identification of action potentials in multi-channel extracellular recordings, in particular from high-density microelectrode arrays with thousands of sensors, is an unresolved problem. While independent component analysis (ICA) achieves rapid unsupervised sorting, it ignores the convolutive structure of extracellular data, thus limiting the unmixing to a subset of neurons. New methodHere we present a spike sorting algorithm based on convolutive ICA (cICA) to retrieve a larger number of accurately sorted neurons than with instantaneous ICA while accounting for signal overlaps. Spike sorting was applied to datasets with varying signal-to-noise ratios (SNR: 3–12) and 27% spike overlaps, sampled at either 11.5 or 23kHz on 4365 electrodes. ResultsWe demonstrate how the instantaneity assumption in ICA-based algorithms has to be relaxed in order to improve the spike sorting performance for high-density microelectrode array recordings. Reformulating the convolutive mixture as an instantaneous mixture by modeling several delayed samples jointly is necessary to increase signal-to-noise ratio. Our results emphasize that different cICA algorithms are not equivalent. Comparison with existing methodsSpike sorting performance was assessed with ground-truth data generated from experimentally derived templates. The presented spike sorter was able to extract ≈90% of the true spike trains with an error rate below 2%. It was superior to two alternative (c)ICA methods (≈80% accurately sorted neurons) and comparable to a supervised sorting. ConclusionOur new algorithm represents a fast solution to overcome the current bottleneck in spike sorting of large datasets generated by simultaneous recording with thousands of electrodes.

Cell type- and activity-dependent extracellular correlates of intracellular spiking.

Despite decades of extracellular action potential (EAP) recordings monitoring brain activity, the biophysical origin and inherent variability of these signals remain enigmatic. We performed whole cell patch recordings of excitatory and inhibitory neurons in rat somatosensory cortex slice while positioning a silicon probe in their vicinity to concurrently record intra- and extracellular voltages for spike frequencies under 20 Hz. We characterize biophysical events and properties (intracellular spiking, extracellular resistivity, temporal jitter, etc.) related to EAP recordings at the single-neuron level in a layer-specific manner. Notably, EAP amplitude was found to decay as the inverse of distance between the soma and the recording electrode with similar (but not identical) resistivity across layers. Furthermore, we assessed a number of EAP features and their variability with spike activity: amplitude (but not temporal) features varied substantially (∼ 30-50% compared with mean) and nonmonotonically as a function of spike frequency and spike order. Such EAP variation only partly reflects intracellular somatic spike variability and points to the plethora of processes contributing to the EAP. Also, we show that the shape of the EAP waveform is qualitatively similar to the negative of the temporal derivative to the intracellular somatic voltage, as expected from theory. Finally, we tested to what extent EAPs can impact the lowpass-filtered part of extracellular recordings, the local field potential (LFP), typically associated with synaptic activity. We found that spiking of excitatory neurons can significantly impact the LFP at frequencies as low as 20 Hz. Our results question the common assertion that the LFP acts as proxy for synaptic activity.

Mapping of Functionally Characterized Cell Classes onto Canonical Circuit Operations in Primate Prefrontal Cortex

Microcircuits are composed of multiple cell classes that likely serve unique circuit operations. But how cell classes map onto circuit functions is largely unknown, particularly for primate prefrontal cortex during actual goal-directed behavior. One difficulty in this quest is to reliably distinguish cell classes in extracellular recordings of action potentials. Here we surmount this issue and report that spike shape and neural firing variability provide reliable markers to segregate seven functional classes of prefrontal cells in macaques engaged in an attention task. We delineate an unbiased clustering protocol that identifies four broad spiking (BS) putative pyramidal cell classes and three narrow spiking (NS) putative inhibitory cell classes dissociated by how sparse, bursty, or regular they fire. We speculate that these functional classes map onto canonical circuit functions. First, two BS classes show sparse, bursty firing, and phase synchronize their spiking to 3-7 Hz (theta) and 12-20 Hz (beta) frequency bands of the local field potential (LFP). These properties make cells flexibly responsive to network activation at varying frequencies. Second, one NS and two BS cell classes show regular firing and higher rate with only marginal synchronization preference. These properties are akin to setting tonically the excitation and inhibition balance. Finally, two NS classes fired irregularly and synchronized to either theta or beta LFP fluctuations, tuning them potentially to frequency-specific subnetworks. These results suggest that a limited set of functional cell classes emerges in macaque prefrontal cortex (PFC) during attentional engagement to not only represent information, but to subserve basic circuit operations.

Nanostructured cavity devices for extracellular stimulation of HL-1 cells.

Microelectrode arrays (MEAs) are state-of-the-art devices for extracellular recording and stimulation on biological tissue. Furthermore, they are a relevant tool for the development of biomedical applications like retina, cochlear and motor prostheses, cardiac pacemakers and drug screening. Hence, research on functional cell-sensor interfaces, as well as the development of new surface structures and modifications for improved electrode characteristics, is a vivid and well established field. However, combining single-cell resolution with sufficient signal coupling remains challenging due to poor cell-electrode sealing. Furthermore, electrodes with diameters below 20 µm often suffer from a high electrical impedance affecting the noise during voltage recordings. In this study, we report on a nanocavity sensor array for voltage-controlled stimulation and extracellular action potential recordings on cellular networks. Nanocavity devices combine the advantages of low-impedance electrodes with small cell-chip interfaces, preserving a high spatial resolution for recording and stimulation. A reservoir between opening aperture and electrode is provided, allowing the cell to access the structure for a tight cell-sensor sealing. We present the well-controlled fabrication process and the effect of cavity formation and electrode patterning on the sensor's impedance. Further, we demonstrate reliable voltage-controlled stimulation using nanostructured cavity devices by capturing the pacemaker of an HL-1 cell network.

Fabrication of MEA‐based nanocavity sensor arrays for extracellular recording of action potentials

Nanocavity sensor arrays are powerful devices for parallel extracellular recordings of action potentials from cell networks. The sensors combine a high spatial resolution and seal resistance between cell and electrode with low electrode impedances, allowing low‐noise electrophysiological measurements of individual cells in a network without the use of CMOS technology. In this paper, we present a new protocol for the simple fabrication of nanocavity sensors, which enables easy modification of standard microelectrode arrays. Our approach enables processing of devices with improved sensor characteristics and flexibility regarding electrode area and aperture size with minimal effort. We characterize the sensors by impedance spectroscopy and demonstrate their applicability by recording action potentials of individual cardiomyocyte‐like cells growing in a network on the chip surface.

The high potency of ME-5343 to aphids is due to a unique mechanism of action

ME-5343 (afidopyropen) is a new and promising insecticide with an unknown mechanism of action that is effective against sucking insects. ME-5343 was highly toxic to pea aphids (Acyrthosiphon pisum), being more toxic than six other widely used insecticides. In contrast, ME-5343 was practically non-toxic to eight other species of insects we tested. ME-5343 was not toxic to German cockroaches (topical application) or American cockroaches (injection), suggesting that lack of toxicity in these species is not due to lack of cuticular penetration. House flies are insensitive to ME-5343 by topical, residual and feeding exposure. Addition of synergists did not change this result, suggesting that insensitivity to ME-5343 in house flies is not due to rapid detoxification nor is it dependent on the method of bioassay used. ME-5343 did not cause firefly lanterns to glow, nor did it prevent the octopamine stimulated lantern glow. Extracellular recordings of action potentials from a tonically active motor nerve of crayfish in situ showed no effects of ME-5343 at concentrations up to 10−5M. These results suggest that the target site of ME-5343 is not the voltage gated sodium channel, voltage gated potassium channel, GABA gated chloride channel, nicotinic acetylcholine receptor, acetylcholinesterase, octopamine receptor or glutamate receptor. ME-5343 injected into crayfish caused flextion of the legs and tail, similar to the symptoms induced by 5-HT (serotonin). We evaluated the effect of ME-5343 on 5-HT2-like receptors with intracellular recordings of excitatory post-synaptic potentials from the peripheral neuromuscular junction of the crayfish and found no effect of ME-5343. Thus, ME-5343 was neither an agonist nor antagonist of 5-HT2 receptors, did not affect neurotransmitter release and did not affect glutamate receptors. We conclude that ME-5343 is highly toxic to aphids and that this is due to a unique, and currently undefined mechanism of action.

Analytic Signal Based Detection of Extracellular Action Potentials

A method for calculating the instantaneous energy of extracellularly recorded action potentials using the analytic signal has been developed and tested. The energy is computed from the recorded signal with Hilbert filter based multi-resolution energy filters with optimized signal to noise ratio. The computed instantaneous energy is used for threshold based detection of action potentials in the signals.

Surface Acoustic Wave Actuated Lab-on-Chip System for Single Cell Analysis

The advent of multi-sensor microelectrodes for extracellular action potential recordings has significantly improved the quality of recorded signals, allowing more reliable detection and classification of action potentials recorded in vivo. These microelectrodes can also be used to localize neuronal signal sources, which may allow experimentalists to estimate other parameters including the neurons’ migration trends, intensities and sizes. This information can also be used to resolve neurons based on their location and type. However, as the exact characteristics of neurons are unknown during in vivo experiments, current attempts to localize neuronal signal sources have not been validated. This article presents experimental validation of a method capable of estimating both the location and intensity of an electrical source. To this end, a stimulating electrode was immersed in a saline solution and its stimulus patterns were recorded by a commercially available four-sensor microelectrode (tetrode). The location of the tetrode was varied with respect to the stimulator, and for each tetrode position, the stimulus was generated at multiple intensity levels. The location and intensity of the source were estimated using the Multiple Signal Classification (MUSIC) algorithm, and the results were quantified by comparison to the parameters’ true values. Localization results, with an accuracy and precision of ~10 μm, and ~11 μm respectively, imply the method’s ability to resolve individual neuronal sources. Similarly, source intensity estimation results indicate that this approach can accurately track changes in neuronal signal amplitude. Together, these results demonstrate the potential of the presented approach in characterizing neuronal signal sources in vivo, which may significantly improve the extracellular recording process and enable a more accurate interpretation of experimental data.

A role for ATP in bronchoconstriction‐induced activation of guinea pig vagal intrapulmonary C‐fibres

Activation of vagal afferent sensory C-fibres in the lungs leads to reflex responses that produce many of the symptoms associated with airway allergy. There are two subtypes of respiratory C-fibres whose cell bodies reside within two distinct ganglia, the nodose and jugular, and whose properties allow for differing responses to stimuli. We here used extracellular recording of action potentials in an ex vivo isolated, perfused lung-nerve preparation to study the electrical activity of nodose C-fibres in response to bronchoconstriction. We found that treatment with both histamine and methacholine caused strong increases in tracheal perfusion pressure that were accompanied by action potential discharge in nodose, but not in jugular C-fibres. Both the increase in tracheal perfusion pressure and action potential discharge in response to histamine were significantly reduced by functionally antagonizing the smooth muscle contraction with isoproterenol, or by blocking myosin light chain kinase with ML-7. We further found that pretreatment with AF-353 or 2',3'-O-(2,4,6-Trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), structurally distinct P2X3 and P2X2/3 purinoceptor antagonists, blocked the bronchoconstriction-induced nodose C-fibre discharge. Likewise, treatment with the ATPase apyrase, in the presence of the adenosine A1 and A2 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH 58261, blocked the C-fibre response to histamine, without inhibiting the bronchoconstriction. These results suggest that ATP released within the tissues in response to bronchoconstriction plays a pivotal role in the mechanical activation of nodose C-fibres.

Effects of barium and ouabain on electrogenesis in various sites of intact and detubulated skeletal muscle fibers of the frog R. temporaria

In loose patch clamp experiments on intact sartorious muscle fibers of the frog Rana temporaria there were revealed two types of wave forms of the extracellularly recorded action potentials (AP). Responses of the first type (T1AP) consisted of the initial positive phase with subsequent phase of strong negativity. Responses of the second type (T2AP) had an additional positive phase concluding their waveform. In the detubulated fibers, only T1AP were recorded. Application Ba2+ (10 microM) to the muscle led to a significant increase in the amplitude of the third T2AP phase, whereas the T1AP characteristics of both intact and detubulated muscle preparations remained unchanged. After the higher applied Ba2+ concentration (50 microM), in some of the studied intact fibers the late positive signal phase was replaced by the late negative phase. The amplitude of the late negative phase was increased markedly by the high-frequency stimulation. At the combined action of ouabain and Ba2+ there was summation of effects. These results can tentatively be explained by that the locally recorded T1AP and T2AP represent responses of the muscle fiber surface and T-tubular membranes and that caracteristics of the late phase of T2AP are determined by activities of T-tubular potassium channels and of Na, K-ATPase.

Influence of unit distance and conduction velocity on the spectra of extracellular action potentials recorded with intrafascicular electrodes

The use of highly selective penetrating electrodes yields multi-unit extracellular action potential (AP) recordings of the nerve fibers in the vicinity of the electrode. Accessing the information carried within the neural data stream further requires discrimination and separation of the multi-unit recording into their constituent multiple single unit spike trains. Shape differences in the single fiber action potentials (SFAPs) are typically used as the criteria for unit separation. The present paper explores the origins of the shape differences through analysis of the SFAP in the frequency domain. We present the derivation and computational model predictions of a method to quantitatively analyse changes in the spectral components of SFAPs with an axially located intrafascicular electrode with non-radially symmetrical sensitivity function. A spatial tissue filter relationship was derived using reciprocity equations in the spatial frequency domain and transformed to time frequency. A three dimensional bioelectrical volume conductor finite element model of a recording electrode residing in a nerve fascicle was developed to explore the potential distribution in the nerve fascicle and further derive the electrode–fiber coupling function in the time-frequency domain. It was found that the spectral distribution of the SFAP was multimodal in nature, similar to empirical reported earlier, and could be predicted by taking the single fiber action currents (SFACs) filtered by the electrode–fiber coupling function. This function manifested itself as a low-pass filter of the SFAC, dependent upon the fiber's location relative to the electrode and conduction velocity. Analysis of the spectral distribution revealed that changes in the landmarks of the distribution could be related to the fiber location and conduction velocity. Moreover, a consistent relationship was found when exploring the distribution of fibers located off the one axis of symmetry.

Frequency-dependent signal transfer at the interface between electrogenic cells and nanocavity electrodes

We present a model to describe the response of chip-based nanocavity sensors during extracellular recording of action potentials. These sensors feature microelectrodes which are embedded in liquid-filled cavities. They can be used for the highly localized detection of electrical signals on a chip. We calculate the sensor's impedance and simulate the propagation of action potentials. Subsequently we apply our findings to analyze cell-chip coupling properties. The results are compared to experimental data obtained from cardiomyocyte-like cells. We show that both the impedance and the modeled action potentials fit the experimental data well. Furthermore, we find evidence for a large seal resistance of cardiomyocytes on nanocavity sensors compared to conventional planar recording systems.

Anaesthetic potency of diazepam is resistant to cholinergic overstimulation

Patients suffering from organophosphorus intoxication are compromised by generalised seizures and respiratory insufficiency, either being potentially lethal. In these patients induction of general anaesthesia to allow artificial ventilation is an important therapeutic option. Previously, it has been demonstrated that cholinergic overstimulation impaired network depressing effects of etomidate and sevoflurane. In this study we tested the impact of cholinergic overstimulation on inhibitory effects of diazepam in organotypic slice cultures of cerebrocortical neurons. Effects of clinically relevant concentrations of diazepam on spontaneous action potential activity were assessed by extracellular action potential recordings under basal cholinergic tone as well as in the presence of acetylcholine (1 μM). Diazepam at anaesthetic concentrations (25–500 μM) impeded spontaneous network activity in a concentration dependent manner (EC 50 80.5 ± 8.0 μM). In the presence of 1 μM acetylcholine the potency of diazepam was not significantly altered (EC 50 83.6 ± 8.4 μM). The results demonstrate that the potency of diazepam to depress neocortical network-excitability is not significantly impaired by cholinergic overstimulation. Diazepam thereby differs from other anaesthetics like etomidate or sevoflurane whose potencies and efficacies were severely attenuated. Hence diazepam might be preferable for induction and maintenance of general anaesthesia in patients suffering from nerve agent intoxication.

A role for ATP in the mechanical activation of nodose C‐fibers in guinea pig lungs

Activation of vagal afferent sensory C-fibers in the lungs leads to reflex responses that produce many of the symptoms associated with airway allergy. There are two subtypes of respiratory C-fibers whose cell bodies reside within two distinct ganglia, the nodose and jugular, and whose properties allow for differing responses to stimuli. We here used extracellular recording of action potentials in an ex vivo isolated, perfused lung-nerve preparation to study the electrical activity of nodose C-fibers in response to mechanical stimulati in guinea pig lung. We used smooth muscle agonists to induce bronchoconstriction, thereby producing a mechanical stimulus, and found that treatment with both histamine (HA; 30 μM; n=15) and methacholine (MCh; 10 μM; n=4) caused strong action potential discharge in nodose C-fibers (10 ± 2 Hz and 15 ± 8 Hz respectively), but not in jugular C-fibers (n=8). Responses to HA were significantly reduced (P<0.05) by blockade of smooth muscle contraction with isoproterenol (100 μM), suggesting that a component of the response is mechanical in nature. The HA-induced activation of the nodose C-fibers was blocked by pretreatment the H1 receptor antagonist pyrilamine (1 μM; n=5). As ATP activation of P2X3 receptors has been found to play a role in mechanosensory transduction, we examined the effect of P2X3 receptor inhibition on the mechanical activation of nodose C-fibers and found that pretreatment with the P2X3 receptor antagonist AF353 (100 μM; n=5) blocked the response to HA. These results suggest that ATP released within the tissues in response to rapid smooth muscle contraction may play a role in the mechanical activation of nodose C-fibers.

Interactions between atropine and etomidate in cortical and spinal networks during cholinergic crisis

General anaesthesia is an important measure in the treatment of patients suffering from organophosphorus intoxication. The intravenous anaesthetic etomidate seems to be well suited for this purpose because of its stable haemodynamics. Here we raise the question whether the anaesthetic properties of etomidate are altered by cholinergic overstimulation and by the cholinergic antagonist atropine. As the neocortex and spinal cord mediate unconsciousness and immobility, organotypic cultures from these regions were used for pharmacological in vitro studies. The effects of etomidate on spontaneous action potential activity were assessed by extracellular action potential recordings under basal cholinergic tone, in the presence of acetylcholine, and in the additional presence of atropine. Etomidate (1 μM) depressed neuronal activity in cortical slices by 47.5 ± 6.9%, but only by 24.9 ± 16.7% in the presence of acetylcholine. If atropine (10 nM) was given in addition, the depression by etomidate was restored (53.4 ± 4.2%). Similar results were obtained using cultured slices from the spinal cord. In summary, cholinergic overstimulation decreased the ability of etomidate to depress neuronal activity in neocortex and spinal cord. Atropine reversed this decrease. Hence, etomidate is potentially applicable for induction of anaesthesia in patients suffering from severe cholinergic crisis, preconditioned that atropine is co-applied.