Simultaneous Electrophysiological Recordings Research Articles

Neural Substrates of Chronic Pain in the Thalamocortical Circuit

AbstractChronic pain (CP), a pathological condition with a large repertory of signs and symptoms, has no recognizable neural functional common hallmark shared by its diverse expressions. The aim of the present research was to identify potential dynamic markers shared in CP models, by using simultaneous electrophysiological extracellular recordings from the rat ventrobasal thalamus and the primary somatosensory cortex. We have been able to extract a neural signature attributable solely to CP, independent from of the originating conditions. This study showed disrupted functional connectivity and increased redundancy in firing patterns in CP models versus controls, and interpreted these signs as a neural signature of CP. In a clinical perspective, we envisage CP as disconnection syndrome and hypothesize potential novel therapeutic appraisal.

A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit

Finding compounds that affect neuronal or muscular function is of great interest as potential therapeutic agents for a variety of neurological disorders. Alternative applications for these compounds include their use as molecular probes as well as insecticides. We have developed a bioassay that requires small amounts of compounds and allows for unbiased screening of biological activity in vivo. For this, we paired administering compounds in a non-invasive manner with simultaneous electrophysiological recordings from a well-characterized neuronal circuit, the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. The circuit encompasses a variety of neurons with cholinergic, glutamatergic, and electrical synapses as well as neuromuscular junctions. Electrophysiological recordings from this system allow for the detection of compound-related effects against any molecular target on these components. Here, we provide evidence that this novel bioassay works with small molecules such as the cholinergic receptor blocker mecamylamine hydrochloride and the potassium channel blocker tetraethylammonium hydroxide, as well as with venom from Conus brunneus and isolated conopeptides. Conopeptides have been developed into powerful drugs, such as the painkillers Prialt™ and Xen2174. However, most conopeptides have yet to be characterized, revealing the need for a rapid and straightforward screening method. Our findings show that mecamylamine hydrochloride, as well as the α-conotoxin ImI, which is known to be an antagonist of the human α7 nicotinic acetylcholine receptor, efficiently disrupted the synaptic transmission of a Drosophila α7 nicotinic acetylcholine receptor-dependent pathway in our circuit but did not affect the function of neurons with other types of synapses. This demonstrates that our bioassay is a valid tool for screening for compounds relevant to human health.

Environmental enrichment differentially modifies specific components of sensory-evoked activity in rat barrel cortex as revealed by simultaneous electrophysiological recordings and optical imaging in vivo

Environmental enrichment of laboratory animals leads to multi-faceted changes to physiology, health and disease prognosis. An important and under-appreciated factor in enhancing cognition through environmental manipulation may be improved basic sensory function. Previous studies have highlighted changes in cortical sensory map plasticity but have used techniques such as electrophysiology, which suffer from poor spatial resolution, or optical imaging of intrinsic signals, which suffers from low temporal resolution. The current study attempts to overcome these limitations by combining voltage-sensitive dye imaging with somatosensory-evoked potential (SEP) recordings: the specific aim was to investigate sensory function in barrel cortex using multi-frequency whisker stimulation under urethane anaesthesia. Three groups of rats were used that each experienced a different level of behavioural or environmental enrichment. We found that enrichment increased all SEP response components subsequent to the initial thalamocortical input, but only when evoked by single stimuli; the thalamocortical component remained unchanged across all animal groups. The optical signal exhibited no changes in amplitude or latency between groups, resembling the thalamocortical component of the SEP response. Permanent and extensive changes to housing conditions conferred no further enhancement to sensory function above that produced by the milder enrichment of regular handling and behavioural testing, a finding with implications for improvements in animal welfare through practical changes to animal husbandry.

Lateral competition for cortical space by layer-specific horizontal circuits

The cerebral cortex constructs a coherent representation of the world by integrating distinct features of the sensory environment. Although these features are processed vertically across cortical layers, horizontal projections interconnecting neighbouring cortical domains allow these features to be processed in a context-dependent manner. Despite the wealth of physiological and psychophysical studies addressing the function of horizontal projections, how they coordinate activity among cortical domains remains poorly understood. We addressed this question by selectively activating horizontal projection neurons in mouse somatosensory cortex, and determined how the resulting spatial pattern of excitation and inhibition affects cortical activity. We found that horizontal projections suppress superficial layers while simultaneously activating deeper cortical output layers. This layer-specific modulation does not result from a spatial separation of excitation and inhibition, but from a layer-specific ratio between these two opposing conductances. Through this mechanism, cortical domains exploit horizontal projections to compete for cortical space.

Loudness Dependence of Auditory Evoked Potentials as Indicator of Central Serotonergic Neurotransmission: Simultaneous Electrophysiological Recordings and In Vivo Microdialysis in the Rat Primary Auditory Cortex

Serotonin released in synapsis is one of the key neurotransmitters in psychiatry and psychopharmacology. The loudness dependence of auditory evoked potentials (LDAEP) has been proposed as a marker for central serotonergic neurotransmission. Several findings in animals and humans support this hypothesis. However, the in vivo measurement of cortical extracellular serotonin levels has never been performed simultaneously with the recording of auditory evoked potentials. The interrelationship between low cortical serotonergic activity and strong LDAEP is yet to be proven. The auditory evoked potentials were recorded in the epidura above the primary auditory cortex of male Wistar rats whereas extracellular serotonin levels in the primary auditory cortex were measured by in vivo microdialysis before and after i.p. application of the selective serotonin reuptake inhibitor citalopram. At baseline, the correlation of coefficients between the LDAEP, especially of the N1 component, and extracellular serotonin levels in the primary auditory cortex was negative. The increase of serotonin levels after citalopram application was significantly related to a decrease of LDAEP of the N1 component (r=-0.86, p=0.003). These data support the view that the LDAEP is closely modulated by cortical serotonergic activity. Thus, the LDAEP might serve as an inversely related marker of synaptically released serotonin in the CNS.

Functional evidence for internal feedback in the songbird brain nucleus HVC

The song control system of songbirds consists mainly of the 'motor pathway' and 'anterior forebrain pathway'. The medial magnocellular nucleus of anterior nidopallium (mMAN) projects to the song control nucleus HVC, which is the point of divergence of the two pathways. We made simultaneous multiunit electrophysiological recordings from the mMAN and HVC in anesthetized Bengalese finches. We confirmed that the mMAN neurons showed song-selective auditory responses, and found temporal correlations between song-related activities of the mMAN and HVC neurons. The temporal relationship between the neural activation of the HVC and mMAN suggests that these nuclei are parts of a closed loop, which could provide internal feedback to the HVC for sequential syllable control.

Imaging of glutamate in brain slices using FRET sensors

The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Főrster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.

Expression of Adhesion Factors Induced by Epileptiform Activity in the Endothelium of the Isolated Guinea Pig Brain In Vitro

Brain inflammation has been recently considered in the pathogenesis of focal epilepsies. Synthesis of pro-inflammatory mediators in the brain was described both in experimental models of seizures and in human postsurgical tissue. Inflammatory mediators may up-regulate endothelial adhesion molecules, therefore promoting adhesion and homing of leucocytes into the brain. In the present study, expression of inducible adhesion factors in brain endothelium was verified after pharmacological induction of seizure-like activity in specific brain areas of the in vitro isolated guinea pig brain. Experiments were performed in isolated guinea-pig brains maintained in vitro by arterial perfusion. In this preparation, brief application of the GABAa receptor-antagonist, bicuculline, consistently induced focal ictal discharges in the limbic region that secondarily diffuse to the neocortex, as verified by simultaneous electrophysiological recording of extracellular activity. At the end of the electrophysiological experiment (after 5 h in vitro), brains were fixed and immunostaining for adhesion molecules P-selectin and ICAM-1 and for Fos protein was evaluated. Immunohistochemical analysis of isolated brains in which seizure-like activity was induced revealed expression of inducible adhesion factors P-selectin and ICAM-1 in the endothelium of small-medium size brain vessels. In particular, the expression of these molecules was consistently observed in all areas involved in epileptic seizure-like ictal activity (limbic cortices and neocortex), and was infrequently found in regions that generated interictal spiking (piriform cortex), suggesting a trigger role played by seizures for endothelial activation. An increase in Fos protein expression was evident in all analyzed limbic areas and in the neocortex, indicating a correlation between the areas of neuronal and endothelial activation. In control brains maintained in vitro for comparable times without induction of epileptiform activity, no immunoreactivity for Fos and adhesion molecules was observed. Seizure-like activity in an in vitro isolated brain preparation induces the expression of adhesion molecules in the cerebral endothelium. These observations indicate that local endothelial activation may represent a crucial step for the development of an inflammatory response induced by seizures, and suggest a possible novel pathogenic mechanism during the process of epileptogenesis.

Persistent activation of protein kinase Cα is not necessary for expression of cerebellar long-term depression

Protein kinase Cα (PKCα) plays a major role in the induction of long-term depression (LTD) in a cerebellar Purkinje cell (PC). The sequential activation model for classical PKC states that PKCα translocates to the plasma membrane by binding Ca ++ and then becomes fully activated by binding diacylglycerol (DAG), which enables estimation of the activity by monitoring its localization. Here, we performed simultaneous electrophysiological recording and fluorescence imaging in a cultured PC expressing GFP-tagged PKCα. When a PC was depolarized, PKCα transiently translocated to the plasma membrane in a Ca ++-dependent manner. Application of membrane permeable DAG or the blocker of DAG lipase prolonged the translocation. These results suggest that the sequential activation model is applicable to PCs. Conjunctive applications of glutamate and depolarization pulse induced LTD, but did not prolong the translocation. Thus, our results imply that persistent activation of PKCα is not necessary for the expression of LTD.

Simple windows-based software for the control of laser scanning confocal microscopes

Rapid advances in computer processing power and the appearance of low cost, high speed multifunction data acquisition hardware makes the control of confocal laser scanning microscopes (CLSMs) with standard laboratory hardware a potentially straightforward task. This paper describes software designed to control a Biorad MRC 600 scan head under Windows 2000 or XP. Using a single high speed, multifunction data acquisition board running under the Igor Pro software environment, waveforms required to drive the scan head galvanometers can be generated and up to two channels of images (768×512 pixels at 8 or 12 bit levels) captured live or at set intervals. Image averaging, zooming, panning and cropping are supported as is live region of interest measurements over time. The software can trigger or be triggered by external devices via TTL signals and, with the addition of a commercial focus controller, Z scans can also be made. Control of the original neutral density and emission filters of multiple laser-based systems is also supported via serial control. The software should be easily adaptable to control custom designed scanning systems or other older makes of CLSM and it can be integrated with additional acquisition boards for simultaneous electrophysiological recording.

Modulation of Serotonergic Neurotransmission by Nitric Oxide

Nitric oxide (NO) and serotonin (5-HT) are two neurotransmitters with important roles in neuromodulation and synaptic plasticity. There is substantial evidence for a morphological and functional overlap between these two neurotransmitter systems, in particular the modulation of 5-HT function by NO. Here we demonstrate for the first time the modulation of an identified serotonergic synapse by NO using the synapse between the cerebral giant cell (CGC) and the B4 neuron within the feeding network of the pond snail Lymnaea stagnalis as a model system. Simultaneous electrophysiological recordings from the pre- and postsynaptic neurons show that blocking endogenous NO production in the intact nervous system significantly reduces the B4 response to CGC activity. The blocking effect is frequency dependent and is strongest at low CGC frequencies. Conversely, bath application of the NO donor DEA/NONOate significantly enhances the CGC-B4 synapse. The modulation of the CGC-B4 synapse is mediated by the soluble guanylate cyclase (sGC)/cGMP pathway as demonstrated by the effects of the sGC antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). NO modulation of the CGC-B4 synapse can be mimicked in cell culture, where application of 5-HT puffs to isolated B4 neurons simulates synaptic 5-HT release. Bath application of diethylamine NONOate (DEA/NONOate) enhances the 5-HT induced response in the isolated B4 neuron. However, the cell culture experiment provided no evidence for endogenous NO production in either the CGC or B4 neuron suggesting that NO is produced by an alternative source. Thus we conclude that NO modulates the serotonergic CGC-B4 synapse by enhancing the postsynaptic 5-HT response.

Grading odor similarities in a Go/No-Go task

Recent studies show that some features of odor perception are predicted by olfactory receptor biophysics and olfactory bulb physiology. Those studies used a behavioral assay in which rodents dig in a dish of scented cage bedding after pretraining to associate a buried reward with an odorant. The advantage of the digging task is an intensity measure of similarity (number of seconds spent digging). The method has the disadvantages of odorant contamination and low control over concentration and timing, making it difficult to use in electrophysiology. We describe an operant task that avoids these disadvantages and provides a reliable intensity-based similarity measure. Odorants can be delivered with a standard air dilution olfactometer, and rats learn to lever press to one odorant and avoid pressing to another in a Go/No-Go (CS+/CS−) task with a partial reinforcement protocol. Generalization tests substitute a portion of the unrewarded CS+ trials with test odorants. The number of generalization trials on which a subject responds to a test odorant is the measure of odor similarity intensity. We present validation tests using mixture component recognition, which show high repeatability, little variability across subjects and no decrease in responding across sessions. The results match those obtained with the digging task in four of five mixtures tested. This method allows optimal control over stimulus parameters and is compatible with simultaneous electrophysiological recording.

Integration of fMRI, NIROT and ERP for studies of human brain function

Different methods of assessing human brain function possess specific advantages and disadvantages compared to others, but it is believed that combining different approaches will provide greater information than can be obtained from each alone. For example, functional magnetic resonance imaging (fMRI) has good spatial resolution but poor temporal resolution, whereas the converse is true for electrophysiological recordings (event-related potentials or ERPs). In this review of recent work, we highlight a novel approach to combining these modalities in a manner designed to increase information on the origins and locations of the generators of specific ERPs and the relationship between fMRI and ERP signals. Near infrared imaging techniques have also been studied as alternatives to fMRI and can be readily integrated with simultaneous electrophysiological recordings. Each of these modalities may in principle be also used in so-called steady-state acquisitions in which the correlational structure of signals from the brain may be analyzed to provide new insights into brain function.

Neuronal basis of optical imaging signals in sensory cortex

The advent of non-invasive imaging methods such as functional magnetic resonance imaging (fMRI) has made it possible to obtain spatial maps of hemodynamic 'activation' in the human brain under a variety of conditions. However, the indirect and poorly understood nature of the coupling between these hemodynamic signals and the underlying neuronal activity has greatly limited the interpretability of neuroimaging results. In our laboratory, we address the question of coupling between pre- and post-synaptic neuronal activity, and the hemodynamic response in rodent somatosensory cortex in response to a localized tactile stimulus. We use full-field multiple-wavelength (spectroscopic) optical imaging of the intrinsic signals that enables simultaneous measurements of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) and simultaneous electrophysiological recordings of spiking and synaptic activity. Our results show that

Simultaneous Electrophysiology and Functional Magnetic Resonance Imaging Studies in Conscious Rats

This chapter discusses the simultaneous electrophysiology and functional magnetic resonance imaging (fMRI) studies in conscious rats. fMRI is a noninvasive procedure for studying the localization of the brain activity and measures the local changes in cerebral hemodynamics. fMRI acquisitions are correlated with spontaneous electrophysiological events, and thus help identify the generators of these events. The evaluation of artifacts due to random motion of the animal during MR data acquisition and between different sampling/acquisitions over a period of time was carried out. It is observed that once the rats are trained successfully, simultaneous electrophysiology and fMRI experiments can be carried out during sleep–wakefulness. The various steps undertaken in order to carry out simultaneous electrophysiological and MRI recordings in the rat brain are explained. It is found that simultaneous electrophysiological recordings and fMRI provide a unique method to study the brain state with good spatial and temporal resolution, but such studies are associated with a number of technical limitations.

Stereotaxic assembly and procedures for simultaneous electrophysiological and MRI study of conscious rat.

A stereotaxic restraining assembly was designed and developed for simultaneous electrophysiological recordings and functional MRI (fMRI) data acquisition from a conscious rat. The design of the nonmagnetic stereotaxic apparatus facilitated the restraining of head and body of the unanesthetized conscious animal during MRI experiments. The apparatus was made of Teflon and Perspex materials with an appropriate size and shape for a 4.7 T / 40 cm animal MRI scanner. Electrodes made from nonmagnetic silver wire were implanted on the skull for recording the electroencephalogram (EEG), the electro-oculogram (EOG), and the electromyogram (EMG), while polycarbonate screws were used for anchoring the electrode assembly. There were no major distortions or artifacts observed in the electrophysiological tracings and MR images. Electrophysiological recordings during fMRI acquisitions are useful to study different neurophysiological mechanisms of sleep and pathophysiology of seizure activity. Integration of electrophysiological recordings (with their good temporal resolution) and MRI (with its superior spatial resolution) is helpful in characterizing the functional state of different brain regions.

Phase differences in electrical discharge rhythms between neuronal populations of the left and right suprachiasmatic nuclei

The circadian pacemaker of the suprachiasmatic nuclei is a complex multioscillator system, which controls circadian and seasonal rhythmicity (Pittendrigh and Daan, 1976; Meijer et al., 1989). A number of clock genes have been identified that play a key role in the generation of circadian rhythms. These clock genes are expressed in a circadian manner as has been shown in mice, rats and hamsters. The time at which their expression reaches peak values differs among the several genes (Lowrey et al., 2000; Daan et al., 2001; Reppert and Weaver, 2001). Expression profiles for a specific gene may also differ among subdivisions of the suprachiasmatic nuclei. It has been shown that mPer1 peaks slightly out of phase in the left and right suprachiasmatic nuclei and that the rhythm in c-fos expression is significantly different between the dorsomedial and ventrolateral regions (Yamazaki et al., 2000; Schwartz et al., 2000). In the special case that the animal shows splitting of its locomotor activity pattern, mPer1 in the left and right suprachiasmatic nuclei appeared to oscillate in antiphase (de la Iglesia et al., 2000). Whether the molecular organization within the suprachiasmatic nuclei plays a role in seasonal rhythmicity, allowing animals to track daylength and become reproductive at the proper phase of the annual cycle, receives increasing interest (Daan et al., 2001; Hastings, 2001). The differences in peak expression times that exist between different genes, and the spatial differences in peak time for single genes, are suggestive of a genetic mechanism underlying the multioscillator structure. It is unknown, however, whether phase differences that are observed at the molecular level exist at the level of electrical activity rhythms in the suprachiasmatic nuclei in order to become potentially functional.In this study we investigated the presence of phase differences in neuronal discharge rhythms in the suprachiasmatic nuclei of the rat. To this purpose we combined simultaneous electrophysiological recordings of neuronal populations in the left and right suprachiasmatic nuclei with a detailed analysis of the phase relationship between them. The results demonstrate that neuronal subpopulations of the suprachiasmatic nuclei show phase differences both in their peak and half-maximum times of up to 4 h. We propose that these phase differences may play a role in the plasticity of the circadian timing system.

Evidence of a neural loop involved in controlling spermathecal contractions in Locusta migratoria

The control of spermathecal contractions in Locusta migratoria via a neural loop was demonstrated using mechanical stimulation and electrophysiological recordings. Extracellular electrophysiological recordings from the receptaculum seminis nerve (N2B2), which innervates the spermathecal sac, were conducted during mechanical stimulation of the genital chamber sensory cells. Activation of the genital chamber sensory cells, using a glass probe approximating the shape and size of an egg, was found to increase the action potential frequency and initiate bursts of action potentials if a tonic frequency of action potentials was present prior to stimulation. If the motor pattern initially consisted of bursts of action potentials, then mechanical stimulation of the genital chamber sensory cells resulted in an increase in firing frequency, in most preparations, with the bursting remaining. Removal of the probe from the genital chamber always returned the motor activity to that noted prior to sensory cell stimulation. Simultaneous electrophysiological recordings from both the left and right receptaculum seminis nerves (N2B2) revealed that the bursts of action potentials were coordinated, although individual action potentials were not coupled one to one. Activation of the genital chamber sensory cells also resulted in increases in spermathecal contraction frequency, an effect which was coordinated with the changes in motor activity. It is proposed that an egg in the genital chamber activates the sensory cells resulting in increases in spermathecal contraction frequency and the subsequent release of spermatozoa onto the micropyle of the egg for fertilization.

Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy

Intracellular Ca 2+ plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca 2+ is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca 2+ dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca 2+ in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37°C using confocal microscopy. The K + salt of the Ca 2+-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca 2+-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca 2+/low-Na + solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 μm from the cut ends. The Ca 2+ signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca 2+ stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37°C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well.

Recruitment of Calcium from Intracellular Stores Does Not Occur during the Expression of Large Spontaneous Calcium Oscillations in GH3 Cells and Lactotropic Cells in Primary Culture

We used simultaneous electrophysiological and intracellular calcium microfluorometry recordings to directly test for the presence of a calcium-induced calcium release mechanism in individual GH₃ cells and cells of a lactotrope-enriched primary culture. In voltage-pulse experiments, extending the duration of a depolarizing voltage-pulse increased intracellular calcium concentration ([Ca²⁺]_i), but we did not observe any evidence for recruitment of intracellular calcium stores. Furthermore, depletion of intracellular calcium stores with thapsigargin or caffeine did not change the calculated calcium buffer capacity of the cells. In current-clamp experiments, we observed elevations in [Ca²⁺]_i in response to spontaneous action potentials. These [Ca²⁺]_i responses were not inhibited by thapsigargin or caffeine. We did find a significant correlation between the magnitude of spontaneous [Ca²⁺]_i increases and action potential duration. We conclude that intracellular calcium stores are not released during the spontaneous [Ca²⁺]_i oscillations observed in these cells, and that the magnitude of [Ca²⁺]_i oscillations is a direct result of extracellular calcium influx that is determined in part by action potential duration.