Extracellularly Recorded Action Potentials Research Articles

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.

Excitatory and Inhibitory Synapses in Neuropeptide Y–Expressing Striatal Interneurons

Although rare, interneurons are pivotal in governing striatal output by extensive axonal arborizations synapsing on medium spiny neurons. Using a genetically modified mouse strain in which a green fluorescent protein (GFP) is driven to be expressed under control of the neuropeptide Y (NPY) promoter, we identified NPY interneurons and compared them with striatal principal neurons. We found that the bacteria artificial chromosome (BAC)-npy mouse expresses GFP with high fidelity in the striatum to the endogenous expression of NPY. Patch-clamp analysis from NPY neurons showed a heterogeneous population of striatal interneurons. In the majority of cells, we observed spontaneous firing of action potentials in extracellular recordings. On membrane rupture, most NPY interneurons could be classified as low-threshold spiking interneurons and had high-input resistance. Voltage-clamp recordings showed that both GABA and glutamate gated ion channels mediate synaptic inputs onto these striatal interneurons. AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) were small in amplitude and infrequent in NPY neurons. Evoked EPSCs did not show short-term plasticity but some rectification. Evoked N-methyl-d-aspartate (NMDA) EPSCs had fast decay kinetics and were poorly sensitive to an NR2B subunit containing NMDA receptor blocker. Spontaneous inhibitory postsynaptic currents (sIPSCs) were mediated by GABA(A) receptors and were quite similar among all striatal neurons studied. On the contrary, evoked IPSCs decayed faster in NPY neurons than in other striatal neurons. These data report for the first time specific properties of synaptic transmission to NPY striatal interneurons.

Substrate arrays of Iridium Oxide microelectrodes for in vitro neuronal interfacing

The design of novel bidirectional interfaces for in vivo and in vitro nervous systems is an important step towards future functional neuroprosthetics. Small electrodes, structures and devices are necessary to achieve high-resolution and target-selectivity during stimulation and recording of neuronal networks, while significant charge transfer and large signal-to-noise ratio are required for accurate time resolution. In addition, the physical properties of the interface should remain stable across time, especially when chronic in vivo applications or in vitro long-term studies are considered, unless a procedure to actively compensate for degradation is provided. In this short report, we describe the use and fabrication of arrays of 120 planar microelectrodes (MEAs) of sputtered Iridium Oxide (IrOx). The effective surface area of individual microelectrodes is significantly increased using electrochemical activation, a procedure that may also be employed to restore the properties of the electrodes as required. The electrode activation results in a very low interface impedance, especially in the lower frequency domain, which was characterized by impedance spectroscopy. The increase in the roughness of the microelectrodes surface was imaged using digital holographic microscopy and electron microscopy. Aging of the activated electrodes was also investigated, comparing storage in saline with storage in air. Demonstration of concept was achieved by recording multiple single-unit spike activity in acute brain slice preparations of rat neocortex. Data suggests that extracellular recording of action potentials can be achieved with planar IrOx MEAs with good signal-to-noise ratios.

TRPA1 in bradykinin-induced mechanical hypersensitivity of vagal C fibers in guinea pig esophagus

Bradykinin (BK) activates sensory nerves and causes hyperalgesia. Transient receptor potential A1 (TRPA1) is expressed in sensory nerves and mediates cold, mechanical, and chemical nociception. TRPA1 can be activated by BK. TRPA1 knockout mice show impaired responses to BK and mechanical nociception. However, direct evidence from sensory nerve terminals is lacking. This study aims to determine the role of TRPA1 in BK-induced visceral mechanical hypersensitivity. Extracellular recordings of action potentials from vagal nodose and jugular neurons are performed in an ex vivo guinea pig esophageal-vagal preparation. Peak frequencies of action potentials of afferent nerves evoked by esophageal distension and chemical perfusion are recorded and compared. BK activates most nodose and all jugular C fibers. This activation is repeatable and associated with a significant increase in response to esophageal distension, which can be prevented by the B2 receptor antagonist WIN64338. TRPA1 agonist allyl isothiocyanate (AITC) activates most BK-positive nodose and jugular C fibers. This is associated with a transient loss of response to mechanical distensions and desensitization to a second AITC perfusion. Desensitization with AITC and pretreatment with TRPA1 inhibitor HC-030031 both inhibit BK-induced mechanical hypersensitivity but do not affect BK-evoked activation in nodose and jugular C fibers. In contrast, esophageal vagal afferent Adelta fibers do not respond to BK or AITC and fail to show mechanical hypersensitivity after BK perfusion. This provides the first evidence directly from visceral sensory afferent nerve terminals that TRPA1 mediates BK-induced mechanical hypersensitivity. This reveals a novel mechanism of visceral peripheral sensitization.

Functionalised microneedles for enhanced neuronal adhesion

Efficient functional coupling of neuronal cells and electronic sensors could result in hybrid bidirectional communication between neurons and computers (L.J. Breckenridge et al., Advantages of using microfabricated extracellular electrodes for in vitro neuronal recordings, J. Neurosci Res. 42 (1995), pp. 266–276; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457–10462). Such systems could enable us to gain insight into the mechanisms of neuro-degenerative diseases like Parkinson's and Alzheimer's disease in vitro or could be used to improve the function and efficiency of devices used in vivo, for example in Deep Brain Stimulation devices that are already used in the treatment of Parkinson's disease. One of the major challenges for the development of reliable neuro-electronic systems is to perform extracellular recordings of action potentials with a high signal-to-noise ratio. The poor quality of these recordings is caused by the culture medium, which is present in the cleft between the cell membrane and the sensor surface (P. Fromherz, Neuroelectronic interfacing: semiconductor chips with ion channels, nerve cells and brain, in Nanoelectronics and Information Technology, R. Waser, ed., Wiley–VCH, Berlin, 2003, pp. 781–810; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457–10462). In this article, we describe a method allowing a reduction of the distance between the membrane and the surface by combining surface chemistry and topography. We have developed a specialised surface chemistry, based on small laminin-derived peptides, which applied onto the topographical structures, triggers their engulfment by the cell membrane in a phagocytosis-like event. In the phagocytotic pit, the distance between the cell membrane and the sensor surface is believed to be minimal. We describe the surface chemistry used for the controlled immobilisation of the small peptides on the surface of the needle-like structures that are manufactured on the surface of electronic devices. PC12 neuro-blastoma cells and genetically-modified HeLa cells have been used to investigate the interaction between the cell membrane and the peptide functionalised topographical structures. The membrane–surface interaction was examined by means of electron microscopy and fluorescence microscopy.

Cortical Networks Grown on Microelectrode Arrays As a Biosensor for Botulinum Toxin

Botulinum toxin (BoNT) is a potent neurotoxin produced by toxigenic strains of Clostridium botulinum. Botulinum toxin poses a major threat since it could be employed in a deliberate attack on the U.S. food supply. Furthermore, BoNT may be liberated in any insufficiently processed food containing a reduced oxygen atmosphere. Hence, rapid and reliable detection of BoNT in foods is necessary to reduce risks posed through food contamination. We present a BoNT biosensor employing living neural cultures grown in vitro on microelectrode arrays (MEAs). An MEA is a culture dish with a grid of electrodes embedded in its surface, enabling extracellular recording of action potentials of neural cultures grown over the array. Pharmaceutical grade BoNT A was applied to the media bath of mature cortical networks cultured on MEAs. Both spontaneous and evoked activities were monitored over 1 wk to quantify changes in the neural population produced by BoNT A. Introduction of BoNT A resulted in an increased duration and number of spikes in spontaneous and evoked bursts relative to control cultures. Increases were significant within 48 h of BoNT A dosage (P < 0.05). Application of BoNT A also induced unique oscillatory behavior within each burst that is reminiscent of early developmental activity patterns rather than the mature cultures used here. Three or more activity peaks were observed in 50% of the BoNT dosed cultures. Control cultures exhibited only a single activity peak. Thus activity of these cortical networks measured with MEAs could provide a valuable substrate for BoNT detection.

A New 3-D Finite-Element Model Based on Thin-Film Approximation for Microelectrode Array Recording of Extracellular Action Potential

A transient finite-element model has been developed to simulate an extracellular action potential recording in a tissue slice by a planar microelectrode array. The thin-film approximation of the active neuron membrane allows the simulation within single finite-element software of the intracellular and extracellular potential fields. In comparison with a compartmental neuron model, it is shown that the thin-film approximation-based model is able to properly represent the neuron bioelectrical behavior in terms of transmembrane current and potential. Moreover, the model is able to simulate extracellular action potential recordings with properties similar to those observed in biological experiments. It is demonstrated that an ideal measurement system model can be used to represent the recording microelectrode, provided that the electronic recording system adapts to the electrode-tissue interface impedance. By comparing it with a point source approximated neuron, it is also shown that the neuron three-dimensional volume should be taken into account to simulate the extracellular action potential recording. Finally, the influence of the electrode size on the signal amplitude is evaluated. This parameter, together with the microelectrode noise, should be taken into account in order to optimize future microelectrode designs in terms of the signal-to-noise ratio.

Symmetric Sensorimotor Somatotopy

BackgroundFunctional imaging has recently been used to investigate detailed somatosensory organization in human cortex. Such studies frequently assume that human cortical areas are only identifiable insofar as they resemble those measured invasively in monkeys. This is true despite the electrophysiological basis of the latter recordings, which are typically extracellular recordings of action potentials from a restricted sample of cells.Methodology/Principal FindingsUsing high-resolution functional magnetic resonance imaging in human subjects, we found a widely distributed cortical response in both primary somatosensory and motor cortex upon pneumatic stimulation of the hairless surface of the thumb, index and ring fingers. Though not organized in a discrete somatotopic fashion, the population activity in response to thumb and index finger stimulation indicated a disproportionate response to fingertip stimulation, and one that was modulated by stimulation direction. Furthermore, the activation was structured with a line of symmetry through the central sulcus reflecting inputs both to primary somatosensory cortex and, precentrally, to primary motor cortex.Conclusions/SignificanceIn considering functional activation that is not somatotopically or anatomically restricted as in monkey electrophysiology studies, our methodology reveals finger-related activation that is not organized in a simple somatotopic manner but is nevertheless as structured as it is widespread. Our findings suggest a striking functional mirroring in cortical areas conventionally ascribed either an input or an output somatotopic function.

Quantifying the isolation quality of extracellularly recorded action potentials

There have been many approaches to the problem of detection and sorting of extra-cellularly recorded action potentials, but only a few methods actually quantify the quality of this fundamental process. In most cases, the quality assessment is based on the subjective judgment of human observers and the recorded units are divided into “well isolated” or “multi-unit” groups. This subjective evaluation precludes comprehensive assessment of single-unit studies since the most basic parameter, i.e. their data quality, is not explicitly defined. Here we propose objective measures to evaluate the quality of spike data, based on the time-stamps of the detected spikes and the high-frequency sampling of the analog signal of cortical and basal-ganglia data. We show that quantification of recording quality by the signal-to-noise ratio (SNR) may be misleading. The recording quality is better assessed by an isolation score that measures the overlap between the noise (non-spike) and the spike clusters. Furthermore, we use a nearest-neighbors algorithm to estimate the proportion of false positive and false negative classification errors. To validate these quality measures, we simulate spike detection and sorting errors and show that the scores are good predictors of the frequency of errors. The reliability of the isolation score is further verified by errors implanted in real basal ganglia data and by using different sorting algorithms. We conclude that quantitative measures of spike isolation can be obtained independently of the method used for spike detection and sorting, and recommend their reports in any study based on the activity of single neurons.

Automated optimal detection and classification of neural action potentials in extra-cellular recordings

Determination of single unit spikes from multiunit spike trains plays a critical role in neurophysiological coding studies which require information about the precise timing of events underlying the neural codes that are the basis of behavior. Searching for optimal spike detection strategies has therefore been the focus of many studies over the past two decades. In this study we describe and implement an algorithm for the optimal real time detection and classification of neural spikes. The algorithm consists of three steps: noise analysis, template generation and real time detection and classification. The first step involves estimating the background noise statistics. In this step, a “cap-fitting” algorithm is used to automatically detect a spike free segment and then the mean, standard deviation and autocorrelation function of the noise are computed. The second step involves generating optimal templates of the spikes from a segment containing both noise and multiunit activity. In this step, a generalized matched filter is used to isolate a set of preliminary spikes from the noise. The first principal component of previously recorded templates is used as the deterministic signal. The preliminary spikes are then clustered in a sub-space spanned by the first three principal components to form new templates. The third step uses these templates for the real time spike detection and classification. In this step the incoming data are projected into a lower dimensional space that is designed to maximally separate the signal from the noise energy. This algorithm provides an accurate estimate of the signal to noise ratio and provides an accurate estimate of spike times and spike shapes.

Using extracellular action potential recordings to constrain compartmental models

We investigate the use of extracellular action potential (EAP) recordings for biophysically faithful compartmental models. We ask whether constraining a model to fit the EAP is superior to matching the intracellular action potential (IAP). In agreement with previous studies, we find that the IAP method under-constrains the parameters. As a result, significantly different sets of parameters can have virtually identical IAP's. In contrast, the EAP method results in a much tighter constraint. We find that the distinguishing characteristics of the waveform--but not its amplitude-resulting from the distribution of active conductances are fairly invariant to changes of electrode position and detailed cellular morphology. Based on these results, we conclude that EAP recordings are an excellent source of data for the purpose of constraining compartmental models.

Online detection and sorting of extracellularly recorded action potentials in human medial temporal lobe recordings, in vivo

Understanding the function of complex cortical circuits requires the simultaneous recording of action potentials from many neurons in awake and behaving animals. Practically, this can be achieved by extracellularly recording from multiple brain sites using single wire electrodes. However, in densely packed neural structures such as the human hippocampus, a single electrode can record the activity of multiple neurons. Thus, analytic techniques that differentiate action potentials of different neurons are required. Offline spike sorting approaches are currently used to detect and sort action potentials after finishing the experiment. Because the opportunities to record from the human brain are relatively rare, it is desirable to analyze large numbers of simultaneous recordings quickly using online sorting and detection algorithms. In this way, the experiment can be optimized for the particular response properties of the recorded neurons. Here we present and evaluate a method that is capable of detecting and sorting extracellular single-wire recordings in realtime. We demonstrate the utility of the method by applying it to an extensive data set we acquired from chronically implanted depth electrodes in the hippocampus of human epilepsy patients. This dataset is particularly challenging because it was recorded in a noisy clinical environment. This method will allow the development of “closed-loop” experiments, which immediately adapt the experimental stimuli and/or tasks to the neural response observed.

Repeated voltage biasing improves unit recordings by reducing resistive tissue impedances

Reactive tissue encapsulation of chronically implanted microelectrode probes can preclude long-term recording of extracellular action potentials. We investigated an intervention strategy for functionally encapsulated microelectrode sites. This method, known as "rejuvenation," involved applying a +1.5 V dc bias to an iridium site for 4 s. Previous studies have demonstrated that rejuvenation resulted in higher signal-to-noise ratios (SNRs) by decreasing noise levels, and reduced 1-kHz site impedances by decreasing the tissue interface resistances. In this study, we have investigated: 1) the duration of a single-voltage bias session and 2) the efficacy of multiple sessions. These questions were addressed through electrophysiological recordings, cyclic voltammetry, and modeling the electrode-tissue interface via an equivalent circuit model fit to impedance spectroscopy data. In the six implants studied, we found SNRs improved for 1-7 days with a peak typically occurring within 24 h of the voltage bias. Root-mean square (RMS) noise of the extracellular recordings decreased for 1-2 days, which paralleled a similar decrease in the adsorbed tissue resistance (Ren) from the model. Implants whose SNR effects lasted more than a day showed stabilized reductions in the extracellular tissue resistance (Rex) and cellular membrane area (Am). Subsequent stimulus sessions were found to drop neural tissue parameters consistently to levels observed immediately after surgery. In most cases, these changes did parallel an improvement in SNR. These findings suggest that rejuvenation may be a useful intervention strategy to prolong the lifetime of chronically implanted microelectrodes.

Optimal filtering for spike sorting of multi-site electrode recordings

We derive an optimal linear filter, to reduce the distortions of the peak amplitudes of action potentials in extracellular multitrode recordings, which are due to background activity and overlapping spikes. This filter is being learned very efficiently from the raw recordings in an unsupervised manner and responds to the average waveform with an impulse of minimal width. The average waveform does not have to be known in advance, but is learned together with the optimal filter. The peak amplitude of a filtered waveform is a more reliable estimate for the amplitude of an action potential than the peak of the biphasic waveform and can improve the accuracy of the event detection and clustering procedures. We demonstrate a spike-sorting application, in which events are detected using the Mahalanobis distance in the N-dimensional space of filtered recordings as a distance measure, and the event amplitudes of the filtered recordings are clustered to assign events to individual units. This method is fast and robust, and we show its performance by applying it to real tetrode recordings of spontaneous activity in the visual cortex of an anaesthetized cat and to realistic artificial data derived therefrom.

An unsupervised automatic method for sorting neuronal spike waveforms in awake and freely moving animals

The present study introduces an approach to automatic classification of extracellularly recorded action potentials of neurons. The classification of spike waveform is considered a pattern recognition problem of special segments of signal that correspond to the appearance of spikes. The spikes generated by one neuron should be recognized as members of the same class. The spike waveforms are described by the nonlinear oscillating model as an ordinary differential equation with perturbation, thus characterizing the signal distortions in both amplitude and phase. It is shown that the use of local variables reduces the problem of spike recognition to the separation of a mixture of normal distributions in the transformed feature space. We have developed an unsupervised iteration-learning algorithm that estimates the number of classes and their centers according to the distance between spike trajectories in phase space. This algorithm scans the learning set to evaluate spike trajectories with maximal probability density in their neighborhood. Following the learning, the procedure of minimal distance is used to perform spike recognition. Estimation of trajectories in phase space requires calculation of the first- and second-order derivatives, and integral operators with piecewise polynomial kernels were used. This provided the computational efficiency of the developed approach for real-time application as required by recordings in behaving animals and in human neurosurgical operations. The new method of spike sorting was tested on simulated and real data and performed better than other approaches currently used in neurophysiology.

Multi-unit spike-triggered averaging: a method for probing the physiology of central synapses

We describe the ongoing development of a method that combines multi-unit extracellular recording with intracellular recording to probe unitary synaptic connections in the central nervous system. In multi-unit spike-triggered averaging (multi-unit STA), intracellular recordings are averaged based on extracellularly recorded action potentials from multiple units to rapidly screen large numbers of neuronal pairs for rare synaptic connections. High throughput is achieved by using many extracellular electrodes and online, automated analysis. Using this approach, we were able to screen dozens of candidate pairs per hour. About 1–2% of these were synaptically connected, and for some of these presynaptic unit isolation was sufficient to accurately quantify synaptic properties such as latency, conductance, kinetics and transmission failure rate. Since it requires only a single intracellular recording, multi-unit STA might be a suitable method for probing unitary synaptic connections in the intact brain, where obtaining multiple intracellular recordings is not feasible.

In vitro networks: cortical mechanisms of anaesthetic action

In vitro networks: cortical mechanisms of anaesthetic action

Airway nerves: in vivo electrophysiology.

Information about the activity of airway sensory afferent nerves in vivo can be obtained electrophysiologically by extracellular recording of action potentials. Apart from data capture, the basic techniques used for recording sensory nerve activity have not advanced greatly in 50 years. However, clearly they continue to contribute vastly to our understanding of the role of these nerves in the control and functions of the airways in disease. This is particularly noticeable with the insight gained into exactly which physiological, biochemical and pharmacological events in the control of lung function rely upon the airway sensory afferent nerves and the subsequent airway vagal reflex arcs.

Hypoxia influences generation and propagation of electrical activity in embryonic cardiomyocyte clusters

The influence of tissue hypoxia on the generation and propagation of excitation was studied in spontaneously beating embryonic cardiomyocyte clusters grown in eight 9–12 days old embryoid bodies. Within the embryoid bodies one to three separately active clusters of cardiomyocytes were found, each having its own pacemaker cell. Lowering of tissue PO 2 caused bradycardia as well as arrhythmia in all embryoid bodies investigated. The mean frequency of the extracellularly recorded action potentials decreased under conditions of pronounced hypoxia from a mean of 1.4–1.8 Hz to below 0.8 Hz. In three embryoid bodies hypoxia-sensitive as well as hypoxia-tolerant cardiomyocyte clusters were found. The hypoxia-insensitive cardiomyocytes showed a low frequency of spontaneous activity. In addition to the observed changes in the generation of excitation, tissue hypoxia caused an approximately 60% reduction in the velocity of conduction within the cardiomyocyte clusters. Moreover, in at least one of the eight experiments propagation failure with an incomplete block in spread of excitation was observed. All hypoxia-induced effects on generation and propagation of embryonic cardiomyocyte excitation were completely reversible after reoxygenation.