Simultaneous Intracellular Recordings Research Articles

Transparent Intracellular Sensing Platform with Si Needles for Simultaneous Live Imaging.

Vertically ordered Si needles are of particular interest for long-term intracellular recording owing to their capacity to infiltrate living cells with negligible damage and minimal toxicity. Such intracellular recordings could greatly benefit from simultaneous live cell imaging without disrupting their culture, contributing to an in-depth understanding of cellular function and activity. However, the use of standard live imaging techniques, such as inverted and confocal microscopy, is currently impeded by the opacity of Si wafers, typically employed for fabricating vertical Si needles. Here, we introduce a transparent intracellular sensing platform that combines vertical Si needles with a percolated network of Au-Ag nanowires on a transparent elastomeric substrate. This sensing platform meets all prerequisites for simultaneous intracellular recording and imaging, including electrochemical impedance, optical transparency, mechanical compliance, and cell viability. Proof-of-concept demonstrations of this sensing platform include monitoring electrical potentials in cardiomyocyte cells and in three-dimensionally engineered cardiovascular tissue, all while conducting live imaging with inverted and confocal microscopes. This sensing platform holds wide-ranging potential applications for intracellular research across various disciplines such as neuroscience, cardiology, muscle physiology, and drug screening.

Calcium transients in intramuscular interstitial cells of Cajal of the murine gastric fundus and their regulation by neuroeffector transmission.

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca2+ and post-junctional electrical responses to neural stimulation. ICC-IM were highly active, generating intracellular Ca2+ -transients that occurred stochastically, from multiple independent sites in single ICC-IM. Ca2+ -transients were not entrained in single ICC-IM or between neighbouring ICC-IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca2+ -transients in ICC-IM. This inhibitory response was often preceded by a summation of Ca2+ -transients that led to a global rise in Ca2+ . Individual ICC-IM responded to nerve stimulation by a global rise in Ca2+ followed by inhibition of Ca2+ -transients. The inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in intracellular Ca2+ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video-imaging revealed that the rise in Ca2+ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca2+ -transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. KEY POINTS: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC-IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs. Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC-IM were examined. ICC-IM were highly active, generating stochastic intracellular Ca2+ -transients. Stimulation of enteric motor nerves abolished Ca2+ -transients in ICC-IM. This inhibitory response was preceded by a global rise in intracellular Ca2+ . Individual ICC-IM responded to nerve stimulation with a rise in Ca2+ followed by inhibition of Ca2+ -transients. Inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in Ca2+ was inhibited by the muscarinic antagonist atropine. Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca2+ and inhibition of Ca2+ -transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials. The data presented support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Cardiotoxicity drug screening based on whole-panel intracellular recording

Unintended binding of small-molecule drugs to ion channels affects electrophysiological properties of cardiomyocytes and potentially leads to arrhythmia and heart failure. The waveforms of intracellular action potentials reflect the coordinated activities of cardiac ion channels and serve as a reliable means for assessing drug toxicity, but the implementation is limited by the low throughput of patch clamp for intracellular recording measurements. In the last decade, several new technologies are being developed to address this challenge. We recently developed the nanocrown electrode array (NcEA) technology that allows robust, parallel, and long-duration recording of intracellular action potentials (iAPs). Here, we demonstrate that NcEAs allow comparison of iAP waveforms before and after drug treatment from the same cell. This self-referencing comparison not only shows distinct drug effects of sodium, potassium, and calcium blockers, but also reveals subtle differences among three subclasses of sodium channel blockers with sub-millisecond accuracy. Furthermore, self-referencing comparison unveils heterogeneous drug responses among different cells. In our study, whole-panel simultaneous intracellular recording can be reliably achieved with ∼94% success rate. The average duration of intracellular recording is ∼30 min and some last longer than 2 h. With its high reliability, long recording duration, and easy-to-use nature, NcEA would be useful for iAP-based preclinical drug screening.

Synchronous inhibitory pathways create both efficiency and diversity in the retina

Sensory receptive fields combine features that originate in different neural pathways. Retinal ganglion cell receptive fields compute intensity changes across space and time using a peripheral region known as the surround, a property that improves information transmission about natural scenes. The visual features that construct this fundamental property have not been quantitatively assigned to specific interneurons. Here, we describe a generalizable approach using simultaneous intracellular and multielectrode recording to directly measure and manipulate the sensory feature conveyed by a neural pathway to a downstream neuron. By directly controlling the gain of individual interneurons in the circuit, we show that rather than transmitting different temporal features, inhibitory horizontal cells and linear amacrine cells synchronously create the linear surround at different spatial scales and that these two components fully account for the surround. By analyzing a large population of ganglion cells, we observe substantial diversity in the relative contribution of amacrine and horizontal cell visual features while still allowing individual cells to increase information transmission under the statistics of natural scenes. Established theories of efficient coding have shown that optimal information transmission under natural scenes allows a diverse set of receptive fields. Our results give a mechanism for this theory, showing how distinct neural pathways synthesize a sensory computation and how this architecture both generates computational diversity and achieves the objective of high information transmission.

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior.

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.

A nanoelectrode array for obtaining intracellular recordings from thousands of connected neurons

Current electrophysiological or optical techniques cannot reliably perform simultaneous intracellular recordings from more than a few tens of neurons. Here, we report a nanoelectrode array that can simultaneously obtain intracellular recordings from thousands of connected mammalian neurons in vitro. The array consists of 4,096 platinum-black electrodes with nanoscale roughness fabricated on top of a silicon chip that monolithically integrates 4,096 microscale amplifiers, configurable into pseudo-current-clamp mode (for concurrent current injection and voltage recording) or into pseudo-voltage-clamp mode (for concurrent voltage application and current recording). We used the array in pseudo-voltage-clamp mode to measure the effects of drugs on ion-channel currents. In pseudo-current-clamp mode, the array recorded intracellular action potentials and post-synaptic potentials from over thousands of neurons. In addition, we mapped over 300 excitatory and inhibitory synaptic connections from over 1,700 neurons that were recorded for 19 mins. This high-throughput intracellular-recording technology could benefit functional connectome mapping, electrophysiological screening, and other functional interrogations of neuronal networks.

Adaptation of Inhibition Mediates Retinal Sensitization

In response to a changing sensory environment, sensory systems adjust their neural code for a number of purposes, including an enhanced sensitivity for novel stimuli, prediction of sensory features, and the maintenance of sensitivity. Retinal sensitization is a form of short-term plasticity that elevates local sensitivity following strong, local, visual stimulation and has been shown to create a prediction of the presence of a nearby localized object. The neural mechanism that generates this elevation in sensitivity remains unknown. Using simultaneous intracellular and multielectrode recording in the salamander retina, we show that a decrease in tonic amacrine transmission is necessary for and is correlated spatially and temporally with ganglion cell sensitization. Furthermore, introducing a decrease in amacrine transmission is sufficient to sensitize nearby ganglion cells. A computational model accounting for adaptive dynamics and nonlinear pathways confirms a decrease in steady inhibitory transmission can cause sensitization. Adaptation of inhibition enhances the sensitivity to the sensory feature conveyed by an inhibitory pathway, creating a prediction of future input.

Membrane Potential Correlates of Network Decorrelation and Improved SNR by Cholinergic Activation in the Somatosensory Cortex.

The nucleus basalis (NB) projects cholinergic axons to the cortex, where they play a major role in arousal, attention, and learning. Cholinergic inputs shift cortical dynamics from synchronous to asynchronous and improve the signal-to-noise ratio (SNR) of sensory responses. However, the underlying mechanisms of these changes remain unclear. Using simultaneous extracellular and whole-cell patch recordings in layer 4 of the mouse barrel cortex, we show that electrical or optogenetic activation of the cholinergic system has a differential effect on ongoing and sensory evoked activities. Cholinergic activation profoundly reduced the large spontaneous fluctuations in membrane potential and decorrelated ongoing activity. However, NB stimulation had no effect on the response to whisker stimulation or on signal correlations. These effects of cholinergic activation provide a unified explanation for the increased SNR of sensory response and for the reduction in noise correlations and explain the shift into the desynchronized cortical state, which are the hallmarks of arousal and attention.SIGNIFICANCE STATEMENT Attention increases the signal-to-noise ratio (SNR) of cortical sensory response, which may reflect either reduction in background firing rate or increased sensory response. Extracellular recordings showed that attention also reduces the correlation in network activity. These effects are partially mediated by cholinergic axons from the nucleus basalis projecting to the entire cortex. To reveal the cellular and synaptic correlates of these cholinergic effects, we performed simultaneous intracellular and LFP recordings in the somatosensory cortex. Global or local cholinergic activation increased the SNR of sensory response mainly by reducing the rate and amplitude of background synaptic activity and also reduced network correlations. Therefore, coding of sensory information is enhanced by the cholinergic system mainly due to a reduction in spontaneous activity.

Effects of ageing on pro-arrhythmic ventricular phenotypes in incrementally paced murine Pgc-1\u03b2\u2212/\u2212 hearts

A range of chronic clinical conditions accompany cardiomyocyte energetic dysfunction and constitute independent risk factors for cardiac arrhythmia. We investigated pro-arrhythmic and arrhythmic phenotypes in energetically deficient C57BL mice with genetic ablation of the mitochondrial promoter peroxisome proliferator-activated receptor-γ coactivator-1β (Pgc-1β), a known model of ventricular arrhythmia. Pro-arrhythmic and cellular action potential (AP) characteristics were compared in intact Langendorff-perfused hearts from young (12–16 week) and aged (> 52 week), wild-type (WT) and Pgc-1β−/− mice. Simultaneous electrocardiographic and intracellular microelectrode recordings were made through successive trains of 100 regular stimuli at progressively incremented heart rates. Aged Pgc-1β−/− hearts displayed an increased incidence of arrhythmia compared to other groups. Young and aged Pgc-1β−/− hearts showed higher incidences of alternans in both AP activation (maximum AP upshoot velocity (dV/dt)max and latency), recovery (action potential duration (APD90) and resting membrane potential (RMP) characteristics compared to WT hearts. This was particularly apparent at lower pacing frequencies. These findings accompanied reduced (dV/dt)max and increased AP latency values in the Pgc-1β−/− hearts. APs observed prior to termination of the protocol showed lower (dV/dt)max and longer AP latencies, but indistinguishable APD90 and RMPs in arrhythmic compared to those in non-arrhythmic hearts. APD restitution analysis showed that Pgc-1β−/− and WT hearts showed similar limiting gradients. However, Pgc-1β−/− hearts had shortened plateau AP wavelengths, particularly in aged Pgc-1β−/− hearts. Pgc-1β−/− hearts therefore show pro-arrhythmic instabilities attributable to altered AP conduction and activation rather than recovery characteristics.

On the Structure of Cortical Microcircuits Inferred from Small Sample Sizes.

The structure in cortical microcircuits deviates from what would be expected in a purely random network, which has been seen as evidence of clustering. To address this issue, we sought to reproduce the nonrandom features of cortical circuits by considering several distinct classes of network topology, including clustered networks, networks with distance-dependent connectivity, and those with broad degree distributions. To our surprise, we found that all of these qualitatively distinct topologies could account equally well for all reported nonrandom features despite being easily distinguishable from one another at the network level. This apparent paradox was a consequence of estimating network properties given only small sample sizes. In other words, networks that differ markedly in their global structure can look quite similar locally. This makes inferring network structure from small sample sizes, a necessity given the technical difficulty inherent in simultaneous intracellular recordings, problematic. We found that a network statistic called the sample degree correlation (SDC) overcomes this difficulty. The SDC depends only on parameters that can be estimated reliably given small sample sizes and is an accurate fingerprint of every topological family. We applied the SDC criterion to data from rat visual and somatosensory cortex and discovered that the connectivity was not consistent with any of these main topological classes. However, we were able to fit the experimental data with a more general network class, of which all previous topologies were special cases. The resulting network topology could be interpreted as a combination of physical spatial dependence and nonspatial, hierarchical clustering.SIGNIFICANCE STATEMENT The connectivity of cortical microcircuits exhibits features that are inconsistent with a simple random network. Here, we show that several classes of network models can account for this nonrandom structure despite qualitative differences in their global properties. This apparent paradox is a consequence of the small numbers of simultaneously recorded neurons in experiment: when inferred via small sample sizes, many networks may be indistinguishable despite being globally distinct. We develop a connectivity measure that successfully classifies networks even when estimated locally with a few neurons at a time. We show that data from rat cortex is consistent with a network in which the likelihood of a connection between neurons depends on spatial distance and on nonspatial, asymmetric clustering.

Modeling extracellular fields for a three-dimensional network of cells using NEURON

BackgroundComputational modeling of biological cells usually ignores their extracellular fields, assuming them to be inconsequential. Though such an assumption might be justified in certain cases, it is debatable for networks of tightly packed cells, such as in the central nervous system and the syncytial tissues of cardiac and smooth muscle. New methodIn the present work, we demonstrate a technique to couple the extracellular fields of individual cells within the NEURON simulation environment. The existing features of the simulator are extended by explicitly defining current balance equations, resulting in the coupling of the extracellular fields of adjacent cells. ResultsWith this technique, we achieved continuity of extracellular space for a network model, thereby allowing the exploration of extracellular interactions computationally. Using a three-dimensional network model, passive and active electrical properties were evaluated under varying levels of extracellular volumes. Simultaneous intracellular and extracellular recordings for synaptic and action potentials were analyzed, and the potential of ephaptic transmission towards functional coupling of cells was explored. Comparison with existing method(s)We have implemented a true bi-domain representation of a network of cells, with the extracellular domain being continuous throughout the entire model. This has hitherto not been achieved using NEURON, or other compartmental modeling platforms. ConclusionsWe have demonstrated the coupling of the extracellular field of every cell in a three-dimensional model to obtain a continuous uniform extracellular space. This technique provides a framework for the investigation of interactions in tightly packed networks of cells via their extracellular fields.

Two Parallel Olfactory Pathways for Processing General Odors in a Cockroach.

In animals, sensory processing via parallel pathways, including the olfactory system, is a common design. However, the mechanisms that parallel pathways use to encode highly complex and dynamic odor signals remain unclear. In the current study, we examined the anatomical and physiological features of parallel olfactory pathways in an evolutionally basal insect, the cockroach Periplaneta americana. In this insect, the entire system for processing general odors, from olfactory sensory neurons to higher brain centers, is anatomically segregated into two parallel pathways. Two separate populations of secondary olfactory neurons, type1 and type2 projection neurons (PNs), with dendrites in distinct glomerular groups relay olfactory signals to segregated areas of higher brain centers. We conducted intracellular recordings, revealing olfactory properties and temporal patterns of both types of PNs. Generally, type1 PNs exhibit higher odor-specificities to nine tested odorants than type2 PNs. Cluster analyses revealed that odor-evoked responses were temporally complex and varied in type1 PNs, while type2 PNs exhibited phasic on-responses with either early or late latencies to an effective odor. The late responses are 30–40 ms later than the early responses. Simultaneous intracellular recordings from two different PNs revealed that a given odor activated both types of PNs with different temporal patterns, and latencies of early and late responses in type2 PNs might be precisely controlled. Our results suggest that the cockroach is equipped with two anatomically and physiologically segregated parallel olfactory pathways, which might employ different neural strategies to encode odor information.

Long-Term Two-Photon Imaging in Awake Macaque Monkey

Successful application of two-photon imaging withgenetic tools in awake macaque monkeys will enable fundamental advances in our understanding of higher cognitive function at the level of molecular and neuronal circuits. Here we report techniques for long-term two-photon imaging in awake macaque monkeys. Using genetically encoded indicators including GCaMP5 and GCaMP6s delivered by AAV2/1 into the visual cortex, we demonstrate that high-quality two-photon imaging of large neuronal populations can be achieved and maintained in awake monkeys for months. Simultaneous intracellular recording and two-photon calcium imaging confirm that fluorescence activity is linearly proportional to neuronal spiking activity across a wide range of firing rates (10Hz to 150Hz). By providing two-photon imaging access to cortical neuronal populations at single-cell or single dendritic spine resolution in awake monkeys, the techniques reported can help bridge the use of modern genetic and molecular tools and the study of higher cognitive function.

Hyperpolarization of Mouse Pulmonary Artery Smooth Muscle and Endothelium by Calcitonin Gene Related Peptide

The goal of these experiments was to develop mouse pulmonary arteries (PAs) as a viable model for investigating the electrophysiological actions of the sensory neurotransmitter calcitonin gene related peptide (CGRP) on membrane potential (Vm) and electrical signal transmission in PA smooth muscle cells (SMCs) and endothelial cells (ECs). Intact PAs were isolated from anesthetized male C57BL/6J mice (3 months old). Immunostaining confirmed that PAs were sparsely innervated by sensory nerves containing CGRP. For intracellular recording, intact PAs (diameter, ~150 μm) were cannulated, pressurized to 16 cm H2O and studied at 37°C. Alternatively, SMCs were gently dissociated to obtain intact endothelial tubes studied at 32°C. We tested the hypothesis that CGRP would hyperpolarize SMCs and ECs in a concentration‐dependent manner. During continuous recording of Vm, increasing [CGRP] from 1 nM to 1 μM hyperpolarized SMCs of intact PAs from −34±2 mV (rest) to −52±3 mV and EC of tubes from −37±4 mV (rest) to −58±4 mV (n ≥ 4 per group); respective EC50 values were 6.2 and 6.8 nM. With dual simultaneous intracellular recording in endothelial tubes, current injection (±0.5 nA) into one EC evoked corresponding Vm responses (±5 mV) in a remote EC (separation distance, 500 μm), confirming electrical signal transmission along PA endothelium. These preliminary studies confirm that CGRP effectively hyperpolarizes SMCs and ECs in mouse PA while validating the utility of this model for investigating the nature of electrical signaling in response to sensory neurotransmission along the PA wall.Support or Funding InformationNIH R37‐HL041026

Dopamine D3 Receptors Inhibit Hippocampal Gamma Oscillations by Disturbing CA3 Pyramidal Cell Firing Synchrony.

Cortical gamma oscillations are associated with cognitive processes and are altered in several neuropsychiatric conditions such as schizophrenia and Alzheimer’s disease. Since dopamine D3 receptors are possible targets in treatment of these conditions, it is of great importance to understand their role in modulation of gamma oscillations. The effect of D3 receptors on gamma oscillations and the underlying cellular mechanisms were investigated by extracellular local field potential and simultaneous intracellular sharp micro-electrode recordings in the CA3 region of the hippocampus in vitro. D3 receptors decreased the power and broadened the bandwidth of gamma oscillations induced by acetylcholine or kainate. Blockade of the D3 receptors resulted in faster synchronization of the oscillations, suggesting that endogenous dopamine in the hippocampus slows down the dynamics of gamma oscillations by activation of D3 receptors. Investigating the underlying cellular mechanisms for these effects showed that D3 receptor activation decreased the rate of action potentials (APs) during gamma oscillations and reduced the precision of the AP phase coupling to the gamma cycle in CA3 pyramidal cells. The results may offer an explanation how selective activation of D3 receptors may impair cognition and how, in converse, D3 antagonists may exert pro-cognitive and antipsychotic effects.

Excitatory connections of nonspiking interneurones in the terminal abdominal ganglion of the crayfish.

The output effects of the nonspiking interneurones in the crayfish terminal abdominal ganglion upon the uropod motor neurones were characterized using simultaneous intracellular recordings. Inhibitory interactions from nonspiking interneurones to the uropod motor neurones were one-way and chemically mediated. The depolarization of the motor neurones with current injection increased the amplitude of the nonspiking interneurone-mediated hyperpolarization, while hyperpolarization of the motor neurone decreased it. By contrast, excitatory interactions from the nonspiking interneurones to the motor neurones were not mediated via chemical synaptic transmissions. These excitatory connections with the slow motor neurones were one-way while connections with fast motor neurones were bidirectional. Nonspiking interneurone-mediated membrane depolarization of the motor neurones was not affected by the passage of hyperpolarizing current. Each motor neurone spike elicited a time-locked EPSP in the nonspiking interneurones with very short delay (0.2ms) that suggested electrical coupling between nonspiking interneurones and motor neurones. Nonspiking interneurones directly control the organization of slow motor neurone activity, while they appear to regulate the background activity of the fast motor neurones. A single nonspiking interneurone is possible to inhibit some inter and/or motor neurones via direct chemical synapses and simultaneously excite other neurones via electrical synapses.

Neuroscience: The cortical connection.

Neurons in the brain's visual cortex receive inputs from thousands of other neurons. But it now emerges that each is strongly connected to only a few others: those most similar to itself. See Letter p.399 The degree to which a neuron influences the activity of others is dependent on the strength of the synaptic connections it makes with its partners, and it is known that this connection strength can vary over two orders of magnitude. Using a combination of two-photon calcium imaging and simultaneous intracellular recordings from pairs of neurons, Thomas Mrsic-Flogel and colleagues show that layer 2/3 neurons in mouse primary visual cortex (V1) follow a simple rule: strong connections are sparse and occur between neurons with correlated responses to visual stimuli, whereas only weak connections link neurons with uncorrelated responses. This bias in functional connection strength may be a means by which neuronal selectivity for visual features is computed in areas downstream of V1.

Human Brain Activity Patterns beyond the Isoelectric Line of Extreme Deep Coma

The electroencephalogram (EEG) reflects brain electrical activity. A flat (isoelectric) EEG, which is usually recorded during very deep coma, is considered to be a turning point between a living brain and a deceased brain. Therefore the isoelectric EEG constitutes, together with evidence of irreversible structural brain damage, one of the criteria for the assessment of brain death. In this study we use EEG recordings for humans on the one hand, and on the other hand double simultaneous intracellular recordings in the cortex and hippocampus, combined with EEG, in cats. They serve to demonstrate that a novel brain phenomenon is observable in both humans and animals during coma that is deeper than the one reflected by the isoelectric EEG, and that this state is characterized by brain activity generated within the hippocampal formation. This new state was induced either by medication applied to postanoxic coma (in human) or by application of high doses of anesthesia (isoflurane in animals) leading to an EEG activity of quasi-rhythmic sharp waves which henceforth we propose to call ν-complexes (Nu-complexes). Using simultaneous intracellular recordings in vivo in the cortex and hippocampus (especially in the CA3 region) we demonstrate that ν-complexes arise in the hippocampus and are subsequently transmitted to the cortex. The genesis of a hippocampal ν-complex depends upon another hippocampal activity, known as ripple activity, which is not overtly detectable at the cortical level. Based on our observations, we propose a scenario of how self-oscillations in hippocampal neurons can lead to a whole brain phenomenon during coma.

Removing bleaching artifacts from voltage sensitive dye recordings with ICA

Voltage sensitive dye (VSD) imaging of multiple neurons becomes one of the most promising up-to-date methods to investigate neuronal network activity. However, optical imaging signals are often superimposed by noise and artifacts. Hence, post-processing methods are needed to overcome this corruption and separate neuronal activity from other signals. One of the significant artifacts in VSD imaging is bleaching, a decrease of the optical signal while the recorded signal of the local field potentials remains unchanged [1]. In this study we used independent component analysis (ICA) in comparison to principal component analysis (PCA) and detrend method to separate the neuronal VSD signals from bleaching artifacts. ICA is a blind source separation method that has been used in many different approaches such as to recover action potentials of neurons in multiple-detector optical recordings [3]. We used the ICA-DTU Toolbox [http://www2.imm.dtu.dk/pubdb/views/publication_details.php?id=4043] with maximum likelihood formulation to identify neuronal signals and the effect of bleaching. Experimentally, we recorded from Retzius cells of an isolated midbody ganglion of the electrophysiologically well- characterized leech nervous system. VSD imaging (Figure A) based on a new VSD dye with a very good signal-to-noise ratio [2] was combined with simultaneous intracellular recording of the membrane potential (Figure B) and electrical stimulation. VSD signals obtained from the region of the Retzius cell body (Figure ​(Figure1A1A and ​and1C,1C, red) and from a slightly larger region (Figure ​(Figure1A1A and ​and1C,1C, blue) were used as input signals to ICA. Of the two components returned by the algorithm one decreased exponentially, while the other resembled neuronal activity. Figure ​Figure1D1D shows an exponential fit of the component representing bleaching (green), and the estimated neuronal activity (red). Taking the lower sampling rate of the VSD signal (100 Hz vs 10,000 Hz) into account, this estimate reflects the dynamics of the intracellular recording (1B) well. While PCA was not able to separate bleaching from neuronal signals (not shown), the detrend method also yielded good results (Figure ​(Figure1E).1E). This method fits a piecewise linear line (1E, green) to the VSD signal, representing bleaching effect. However, the difference (1E, red) between the original trace and this line represents the graded de-and hyperpolarisations of the membrane potential seen in the intracellular recording (1B) less well than the ICA result (1D, red). Figure 1 VSD and intracellular recording of leech ganglion and analysis results.

Optopatcher—An electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation

Optogenetics has rapidly become a standard method in neuroscience research. Although significant progress has been made in the development of molecular tools, refined techniques for combined light delivery and recording in vivo are still lacking. For example, simultaneous intracellular recording and light stimulation have only been possible by using two separate positioning systems. To overcome this limitation, we have developed a glass pipette holder which contains an additional port for the insertion of an optical fiber into the pipette. This device, which we called “optopatcher” allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. In addition, replacement of standard pipettes is done as easily as with the available commercial holders. Here we used the optopatcher in vivo to record the membrane potential of neurons from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter. We demonstrate the utility of the optopatcher by recording LFP and intracellular responses to light stimulation.