Spatiotemporal structure of sensory-evoked and spontaneous activity revealed by mesoscale imaging in anesthetized and awake mice.

Stimuli-evoked and spontaneous brain activity propagates across the cortex in diverse spatiotemporal patterns. Despite extensive studies, the relationship between spontaneous and evoked activity is poorly understood. We investigate this relationship by comparing the amplitude, speed, direction, and complexity of propagation trajectories of spontaneous and evoked activity elicited with visual, auditory, and tactile stimuli using mesoscale wide-field imaging in mice. For both spontaneous and evoked activity, the speed and direction of propagation is modulated by the amplitude. However, spontaneous activity has a higher complexity of the propagation trajectories. For low stimulus strengths, evoked activity amplitude and speed is similar to that of spontaneous activity but becomes dissimilar at higher stimulus strengths. These findings are consistent with observations that primary sensory areas receive widespread inputs from other cortical regions, and during rest, the cortex tends to reactivate traces of complex multisensory experiences that might have occurred in exhibition of different behaviors.

Enabling comprehensive optogenetic studies of mouse hearts by simultaneous opto-electrical panoramic mapping and stimulation.

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.

Idebenone Protects against Atherosclerosis in Apolipoprotein E-Deficient Mice Via Activation of the SIRT3-SOD2-mtROS Pathway.

PURPOSE: Atherosclerosis, a chronic disease of the arteries, results from pathological processes including the accumulation and aggregation of oxidized low-density lipoprotein (oxLDL) in the vessel walls, development of neointima, formation of a fibrous cap, and migration of immune cells to damaged vascular endothelium. Recent studies have shown that mitochondrial dysfunction is closely associated with the development and progression of atherosclerosis. Idebenone, a short-chain benzoquinone similar in structure to coenzyme Q10, can effectively clear oxygen free radicals as an electron carrier and antioxidant. In the present study, we aim to investigate weather idebenone protects against atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. METHODS: apoE-/- mice receiving a high-fat diet (HFD) were treated with idebenone for 16 weeks. A total of 60 mice were randomized into the following four groups: (1) HFD, (2) HFD and low-dose idebenone (100 mg/kg/d), (3) HFD and medium-dose idebenone (200 mg/kg/d), and (4) HFD and high-dose (400 mg/kg/d). Proteomic analysis was performed between the HFD and idebenone-high-dose group. Plaque analysis was carried out by histological and immunohistochemical staining. Western blot, TUNEL staining, and MitoSOX assays were performed in human umbilical vein endothelial cells (HUVECs) to investigate the SIRT3-SOD2-mtROS pathway. RESULTS: Histological and morphological analysis demonstrated that idebenone significantly reduced plaque burden and plaque size. Idebenone treatment effectively stabilized the atherosclerotic plaques. In mice treated with idebenone, 351 up-regulated and 379 down-regulated proteins were found to be significantly altered in proteomic analysis. In particular, the expression of SIRT3, SOD2, and NLRP3 was significantly regulated in the idebenone treatment groups compared with the HFD group both in vivo and in vitro. We further confirmed that idebenone protected against endothelial cell damage and inhibited the production of mitochondrial reactive oxygen species (mtROS) in cholesterol-treated HUVECs. CONCLUSIONS: We demonstrated that idebenone acted as a mitochondrial protective agent by inhibiting the activation of NLPR3 via the SIRT3-SOD2-mtROS pathway. Idebenone may be a promising therapy for patients with atherosclerosis by improving mitochondrial dysfunction and inhibiting oxidative stress.

Optical Mapping-Validated Machine Learning Improves Atrial Fibrillation Driver Detection by Multi-Electrode Mapping.

BACKGROUND: Atrial fibrillation (AF) can be maintained by localized intramural reentrant drivers. However, AF driver detection by clinical surface-only multielectrode mapping (MEM) has relied on subjective interpretation of activation maps. We hypothesized that application of machine learning to electrogram frequency spectra may accurately automate driver detection by MEM and add some objectivity to the interpretation of MEM findings. METHODS: Temporally and spatially stable single AF drivers were mapped simultaneously in explanted human atria (n=11) by subsurface near-infrared optical mapping (NIOM; 0.3 mm2 resolution) and 64-electrode MEM (higher density or lower density with 3 and 9 mm2 resolution, respectively). Unipolar MEM and NIOM recordings were processed by Fourier transform analysis into 28 407 total Fourier spectra. Thirty-five features for machine learning were extracted from each Fourier spectrum. RESULTS: Targeted driver ablation and NIOM activation maps efficiently defined the center and periphery of AF driver preferential tracks and provided validated annotations for driver versus nondriver electrodes in MEM arrays. Compared with analysis of single electrogram frequency features, averaging the features from each of the 8 neighboring electrodes, significantly improved classification of AF driver electrograms. The classification metrics increased when less strict annotation, including driver periphery electrodes, were added to driver center annotation. Notably, f1-score for the binary classification of higher-density catheter data set was significantly higher than that of lower-density catheter (0.81±0.02 versus 0.66±0.04, P<0.05). The trained algorithm correctly highlighted 86% of driver regions with higher density but only 80% with lower-density MEM arrays (81% for lower-density+higher-density arrays together). CONCLUSIONS: The machine learning model pretrained on Fourier spectrum features allows efficient classification of electrograms recordings as AF driver or nondriver compared with the NIOM gold-standard. Future application of NIOM-validated machine learning approach may improve the accuracy of AF driver detection for targeted ablation treatment in patients.

Validation of Intraoperative Catheter Phase Mapping Using a Simultaneous Optical Measurement System in Rabbit Ventricular Myocardium.

BACKGROUND: Recently, an interoperative catheter electrode mapping system, termed ExTRa Mapping (EXT), was developed for precise diagnosis and effective treatment of non-paroxysmal atrial fibrillations (non-PAF). However, the mapping accuracy of EXT is still unclear.Methods and Results:In this study, the reliability of the EXT in comparison with that of high-resolution optical membrane potential mapping was compared. Spiral wave re-entries (SWRs) were induced in the excised rabbit hearts (n=8, 42 episodes). Electrical signals were measured by electrodes on a transparent silicone plate, with the same arrangement as in the clinical catheter, and fluorescence signals were recorded simultaneously across the plate. Based on the phase maps derived by EXT, activation patterns (one-directed propagations: 26, rotational activities: 16) were identified correctly with 95% accuracy (40/42), and the correlation coefficient of the ratio of the non-passive period was 0.95. In the rotational episodes (15), the mean position error of the centers of gravity of the SWR trajectory (2,000 ms) was 2.0 mm. For the one-directional episodes (25), the correlation coefficient of the directions of one-way propagation was 0.99. CONCLUSIONS: The phase map sequence by EXT is consistent with that by the analyses of high-resolution optical mapping. EXT is reliable for analyzing the activation pattern in the region of interest.

Differential cortical and subcortical projection targets of subfields in the core region of mouse auditory cortex.

The core region of the rodent auditory cortex has two areas: the primary auditory area (A1) and the anterior auditory field (AAF). However, the functional difference between these areas is unclear. To elucidate this issue, here we studied the projections from A1 and AAF in mice using adeno-associated virus (AAV) vectors expressing either a green fluorescent protein or a red fluorescent protein. After mapping A1 and AAF using optical imaging, we injected a distinct AAV vector into each of the two fields at a frequency-matched high-frequency location. We found that A1 and AAF projected commonly to virtually all target areas examined, but each field had its own preference for projection targets. Frontal and parietal regions were the major cortical targets: in the frontal cortex, A1 and AAF showed dominant projections to the anterior cingulate cortex Cg1 and the secondary motor cortex (M2), respectively; in the parietal cortex, A1 and AAF exhibited dense projections to the medial secondary visual cortex and the posterior parietal cortex (PPC), respectively. Although M2 and PPC received considerable input from A1 as well, A1 innervated the medial part whereas AAF innervated the lateral part of these cortical regions. A1 also projected to the orbitofrontal cortex, while AAF also projected to the primary somatosensory cortex and insular auditory cortex. As for subcortical projections, A1 and AAF projected to a common ventromedial region in the caudal striatum with a comparable strength; they also both projected to the medial geniculate body and the inferior colliculus, innervating common and distinct divisions of the nuclei. A1 also projected to visual subcortical structures, such as the superior colliculus and the lateral posterior nucleus of the thalamus, where fibres from AAF were sparse. Our results demonstrate the preference of A1 and AAF for cortical and subcortical targets, and for divisions in individual target. The preference of A1 and AAF for sensory-related structures suggest a role for A1 in providing auditory information for audio-visual association at both the cortical and subcortical level, and a distinct role of AAF in providing auditory information for association with somatomotor information in the cortex.

Development, in vitro validation and human application of a novel method to identify arrhythmia mechanisms: The stochastic trajectory analysis of ranked signals mapping method.

INTRODUCTION: Stochastic trajectory analysis of ranked signals (STAR) is a novel method for mapping arrhythmia. The aim was to describe its development and validation as a mapping tool. METHODS AND RESULTS: The method ranks electrodes in terms of the proportion of the time they lead relative to neighboring electrodes and ascribes a predominant direction of activation between electrodes. This was conceived with the aim of mapping atrial fibrillation (AF) drivers. Validation of this approach was performed in stages. First, in vitro simultaneous multi-electrode array and optical mapping were performed on spontaneously fibrillating HL1 cell cultures, to determine if such a method would be able to determine early sites of activation (ESA). A clinical study acquiring unipolar electrograms using a 64-pole basket for the purposes of STAR mapping in patients undergoing atrial tachycardia (AT) ablation. STAR maps were analyzed by physicians to see if arrhythmia mechanisms could be correctly determined. Mapping was then repeated during atrial pacing. STAR mapping of in vitro activation sequences accurately correlated to the optical maps of planar and rotational activation. Thirty-two ATs were mapped in 25 patients. The ESA accurately identified focal/micro-reentrant ATs and the mechanism of macro-reentrant ATs was effectively demonstrated. STAR method accurately identified four pacing sites in all patients. CONCLUSIONS: This novel STAR method correlated well with the gold standard of optical mapping in vitro and was able to accurately identify AT mechanisms. Further analysis is needed to determine whether the method might be of use mapping AF.

In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models.

AIMS: Cardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models. METHODS AND RESULTS: First, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63-138) cm/s vs. 51 (41-64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217-254) ms vs. 192 (182-233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min-1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA. CONCLUSIONS: In vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.

Compartmentalized Structure of the Moderator Band Provides a Unique Substrate for Macroreentrant Ventricular Tachycardia.

Background Papillary muscles are an important source of ventricular tachycardia (VT). Yet little is known about the role of the right ventricular (RV) endocavity structure, the moderator band (MB). The aim of this study was to determine the characteristics of the MB that may predispose to arrhythmia substrates. Methods Ventricular wedge preparations with intact MBs were studied from humans (n=2) and sheep (n=15; 40-50 kg). RV endocardium was optically mapped, and electrical recordings were measured along the MB and septum. S1S2 pacing of the RV free wall, MB, or combined S1-RV S2-MB sites were assessed. Human (n=2) and sheep (n=4) MB tissue constituents were assessed histologically. Results The MB structure was remarkably organized as 2 excitable, yet uncoupled compartments of myocardium and Purkinje. In humans, action potential duration heterogeneity between MB and RV myocardium was found (324.6±12.0 versus 364.0±8.4 ms; P<0.0001). S1S2-MB pacing induced unidirectional propagation via MB myocardium, permitting sustained macroreentrant VT. In sheep, the incidence of VT for RV, MB, and S1-RV S2-MB pacing was 1.3%, 5.1%, and 10.3%. Severing the MB led to VT termination, confirming a primary arrhythmic role. Inducible preparations had shorter action potential duration in the MB than RV (259.3±45.2 versus 300.7±38.5 ms; P<0.05), whereas noninducible preparations showed no difference (312.0±30.3 versus 310.0±24.6 ms, respectively). Conclusions The MB presents anatomic and electrical compartmentalization between myocardium and Purkinje fibers, providing a substrate for macroreentry. The vulnerability to sustain VT via this mechanism is dependent on MB structure and action potential duration gradients between the RV free wall and MB.

Organization of auditory areas in the superior temporal gyrus of marmoset monkeys revealed by real-time optical imaging.

The prevailing model of the primate auditory cortex proposes a core-belt-parabelt structure. The model proposes three auditory areas in the lateral belt region; however, it may contain more, as this region has been mapped only at a limited spatial resolution. To explore this possibility, we examined the auditory areas in the lateral belt region of the marmoset using a high-resolution optical imaging technique. Based on responses to pure tones, we identified multiple areas in the superior temporal gyrus. The three areas in the core region, the primary area (A1), the rostral area (R), and the rostrotemporal area, were readily identified from their frequency gradients and positions immediately ventral to the lateral sulcus. Three belt areas were identified with frequency gradients and relative positions to A1 and R that were in agreement with previous studies: the caudolateral area, the middle lateral area, and the anterolateral area (AL). Situated between R and AL, however, we identified two additional areas. The first was located caudoventral to R with a frequency gradient in the ventrocaudal direction, which we named the medial anterolateral (MAL) area. The second was a small area with no obvious tonotopy (NT), positioned between the MAL and AL areas. Both the MAL and NT areas responded to a wide range of frequencies (at least 2-24 kHz). Our results suggest that the belt region caudoventral to R is more complex than previously proposed, and we thus call for a refinement of the current primate auditory cortex model.

Local and Global Spatial Organization of Interaural Level Difference and Frequency Preferences in Auditory Cortex.

Despite decades of microelectrode recordings, fundamental questions remain about how auditory cortex represents sound-source location. Here, we used in vivo 2-photon calcium imaging to measure the sensitivity of layer II/III neurons in mouse primary auditory cortex (A1) to interaural level differences (ILDs), the principal spatial cue in this species. Although most ILD-sensitive neurons preferred ILDs favoring the contralateral ear, neurons with either midline or ipsilateral preferences were also present. An opponent-channel decoder accurately classified ILDs using the difference in responses between populations of neurons that preferred contralateral-ear-greater and ipsilateral-ear-greater stimuli. We also examined the spatial organization of binaural tuning properties across the imaged neurons with unprecedented resolution. Neurons driven exclusively by contralateral ear stimuli or by binaural stimulation occasionally formed local clusters, but their binaural categories and ILD preferences were not spatially organized on a more global scale. In contrast, the sound frequency preferences of most neurons within local cortical regions fell within a restricted frequency range, and a tonotopic gradient was observed across the cortical surface of individual mice. These results indicate that the representation of ILDs in mouse A1 is comparable to that of most other mammalian species, and appears to lack systematic or consistent spatial order.

A double-sided microscope to realize whole-ganglion imaging of membrane potential in the medicinal leech.

Studies of neuronal network emergence during sensory processing and motor control are greatly facilitated by technologies that allow us to simultaneously record the membrane potential dynamics of a large population of neurons in single cell resolution. To achieve whole-brain recording with the ability to detect both small synaptic potentials and action potentials, we developed a voltage-sensitive dye (VSD) imaging technique based on a double-sided microscope that can image two sides of a nervous system simultaneously. We applied this system to the segmental ganglia of the medicinal leech. Double-sided VSD imaging enabled simultaneous recording of membrane potential events from almost all of the identifiable neurons. Using data obtained from double-sided VSD imaging, we analyzed neuronal dynamics in both sensory processing and generation of behavior and constructed functional maps for identification of neurons contributing to these processes.

An operant-based detection method for inferring tinnitus in mice.

BACKGROUND: Subjective tinnitus is a hearing disorder in which a person perceives sound when no external sound is present. It can be acute or chronic. Because our current understanding of its pathology is incomplete, no effective cures have yet been established. Mouse models are useful for studying the pathophysiology of tinnitus as well as for developing therapeutic treatments. NEW METHOD: We have developed a new method for determining acute and chronic tinnitus in mice, called sound-based avoidance detection (SBAD). The SBAD method utilizes one paradigm to detect tinnitus and another paradigm to monitor possible confounding factors, such as motor impairment, loss of motivation, and deficits in learning and memory. RESULTS: The SBAD method has succeeded in monitoring both acute and chronic tinnitus in mice. Its detection ability is further validated by functional studies demonstrating an abnormal increase in neuronal activity in the inferior colliculus of mice that had previously been identified as having tinnitus by the SBAD method. COMPARISON WITH EXISTING METHODS: The SBAD method provides a new means by which investigators can detect tinnitus in a single mouse accurately and with more control over potential confounding factors than existing methods. CONCLUSION: This work establishes a new behavioral method for detecting tinnitus in mice. The detection outcome is consistent with functional validation. One key advantage of mouse models is they provide researchers the opportunity to utilize an extensive array of genetic tools. This new method could lead to a deeper understanding of the molecular pathways underlying tinnitus pathology.

Differential Covariance: A New Class of Methods to Estimate Sparse Connectivity from Neural Recordings.

With our ability to record more neurons simultaneously, making sense of these data is a challenge. Functional connectivity is one popular way to study the relationship of multiple neural signals. Correlation-based methods are a set of currently well-used techniques for functional connectivity estimation. However, due to explaining away and unobserved common inputs (Stevenson, Rebesco, Miller, & Körding, 2008 ), they produce spurious connections. The general linear model (GLM), which models spike trains as Poisson processes (Okatan, Wilson, & Brown, 2005 ; Truccolo, Eden, Fellows, Donoghue, & Brown, 2005 ; Pillow et al., 2008 ), avoids these confounds. We develop here a new class of methods by using differential signals based on simulated intracellular voltage recordings. It is equivalent to a regularized AR(2) model. We also expand the method to simulated local field potential recordings and calcium imaging. In all of our simulated data, the differential covariance-based methods achieved performance better than or similar to the GLM method and required fewer data samples. This new class of methods provides alternative ways to analyze neural signals.

Optical Mapping Approaches on Muscleblind-Like Compound Knockout Mice for Understanding Mechanistic Insights Into Ventricular Arrhythmias in Myotonic Dystrophy.

BACKGROUND: Cardiac arrhythmias are common causes of death in patients with myotonic dystrophy (dystrophia myotonica [DM]). Evidence shows that atrial tachyarrhythmia is an independent risk factor for sudden death; however, the relationship is unclear. METHODS AND RESULTS: Control wild-type (Mbnl1+/+; Mbnl2+/+ ) and DM mutant (Mbnl1-/-; Mbnl2+/- ) mice were generated by crossing double heterozygous knockout (Mbnl1+/-; Mbnl2+/- ) mice. In vivo electrophysiological study and optical mapping technique were performed to investigate mechanisms of ventricular tachyarrhythmias. Transmission electron microscopy scanning was performed for myocardium ultrastructural analysis. DM mutant mice were more vulnerable to anesthesia medications and program electrical pacing: 2 of 12 mice had sudden apnea and cardiac arrest during premedication of general anesthesia; 9 of the remaining 10 had atrial tachycardia and/or atrioventricular block, but none of the wild-type mice had spontaneous arrhythmias; and 9 of 10 mice had pacing-induced ventricular tachyarrhythmias, but only 1 of 14 of the wild-type mice. Optical mapping studies revealed prolonged action potential duration, slower conduction velocity, and steeper conduction velocity restitution curves in the DM mutant mice than in the wild-type group. Spatially discordant alternans was more easily inducible in DM mutant than wild-type mice. Transmission electron microscopy showed disarranged myofibrils with enlarged vacuole-occupying mitochondria in the DM mutant group. CONCLUSIONS: This DM mutant mouse model presented with clinical myofibril ultrastructural abnormality and cardiac arrhythmias, including atrial tachyarrhythmias, atrioventricular block, and ventricular tachyarrhythmias. Optical mapping studies revealed prolonged action potential duration and slow conduction velocity in the DM mice, leading to vulnerability of spatially discordant alternans and ventricular arrhythmia induction to pacing.

BK Channels Mediate Synaptic Plasticity Underlying Habituation in Rats.

Habituation is a basic form of implicit learning and represents a sensory filter that is disrupted in autism, schizophrenia, and several other mental disorders. Despite extensive research in the past decades on habituation of startle and other escape responses, the underlying neural mechanisms are still not fully understood. There is evidence from previous studies indicating that BK channels might play a critical role in habituation. We here used a wide array of approaches to test this hypothesis. We show that BK channel activation and subsequent phosphorylation of these channels are essential for synaptic depression presumably underlying startle habituation in rats, using patch-clamp recordings and voltage-sensitive dye imaging in slices. Furthermore, positive modulation of BK channels in vivo can enhance short-term habituation. Although results using different approaches do not always perfectly align, together they provide convincing evidence for a crucial role of BK channel phosphorylation in synaptic depression underlying short-term habituation of startle. We also show that this mechanism can be targeted to enhance short-term habituation and therefore to potentially ameliorate sensory filtering deficits associated with psychiatric disorders.SIGNIFICANCE STATEMENT Short-term habituation is the most fundamental form of implicit learning. Habituation also represents a filter for inundating sensory information, which is disrupted in autism, schizophrenia, and other psychiatric disorders. Habituation has been studied in different organisms and behavioral models and is thought to be caused by synaptic depression in respective pathways. The underlying molecular mechanisms, however, are poorly understood. We here identify, for the first time, a BK channel-dependent molecular synaptic mechanism leading to synaptic depression that is crucial for habituation, and we discuss the significance of our findings for potential treatments enhancing habituation.

An improved method for growing neurons: Comparison with standard protocols.

BACKGROUND: Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. NEW METHOD: Our culturing protocol is based on a novel serum-free preparation of astrocyte - conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). RESULTS: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. COMPARISON WITH EXISTING METHOD(S): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. CONCLUSIONS: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.

Effect of spatial resolution and filtering on mapping cardiac fibrillation.

BACKGROUND: Endocardial mapping tools use variable interelectrode resolution, whereas body surface mapping tools use narrow bandpass filtering (BPF) to map fibrillatory mechanisms established by high-resolution optical imaging. OBJECTIVE: The purpose of this study was to study the effect of resolution and BPF on the underlying mechanism being mapped. METHODS: Hearts from 14 healthy New Zealand white rabbits were Langendorff perfused. We studied the effect of spatial resolution and BPF on the location and characterization of rotors by comparing phase singularities detected by high-resolution unfiltered optical maps and of fibrillating myocardium with decimated and filtered maps with simulated electrode spacing of 2, 5, and 8 mm. RESULTS: As we decimated the maps with 2-mm, 5-mm, and 8-mm interelectrode spacing, the mean ( ± SD) number of rotors detected decreased from 10.2 ± 9.6, 1.6 ± 3.2, and 0.2 ± 0.5, respectively. Lowering the resolution led to synthesized pseudo-rotors that may be inappropriately identified. Applying a BPF led to fewer mean phase singularities detected (248 ± 207 vs 333 ± 130; P<.01), giving the appearance of pseudo-spatial stability measured as translation index (with BPF 3.6 ± 0.4 mm vs 4.0 ± 0.5 mm without BPF; P<.01) and pseudo-temporal stability with longer duration (70.0 ± 17.6 ms in BPF maps vs 44.1 ± 6.6 ms in unfiltered maps; P<.001) than true underlying fibrillating myocardium mapped. CONCLUSION: Electrode resolution and BPF of electrograms can result in distortion of the underlying electrophysiology of fibrillation. Newer mapping techniques need to demonstrate sensitivity analysis to quantify the degree of distortion before clinical use to avoid inaccurate electrophysiologic interpretation.

Thalamic state control of cortical paired-pulse dynamics.

Sensory stimulation drives complex interactions across neural circuits as information is encoded and then transmitted from one brain region to the next. In the highly interconnected thalamocortical circuit, these complex interactions elicit repeatable neural dynamics in response to temporal patterns of stimuli that provide insight into the circuit properties that generated them. Here, using a combination of in vivo voltage-sensitive dye (VSD) imaging of cortex, single-unit recording in thalamus, and optogenetics to manipulate thalamic state in the rodent vibrissa pathway, we probed the thalamocortical circuit with simple temporal patterns of stimuli delivered either to the whiskers on the face (sensory stimulation) or to the thalamus directly via electrical or optogenetic inputs (artificial stimulation). VSD imaging of cortex in response to whisker stimulation revealed classical suppressive dynamics, while artificial stimulation of thalamus produced an additional facilitation dynamic in cortex not observed with sensory stimulation. Thalamic neurons showed enhanced bursting activity in response to artificial stimulation, suggesting that bursting dynamics may underlie the facilitation mechanism we observed in cortex. To test this experimentally, we directly depolarized the thalamus, using optogenetic modulation of the firing activity to shift from a burst to a tonic mode. In the optogenetically depolarized thalamic state, the cortical facilitation dynamic was completely abolished. Together, the results obtained here from simple probes suggest that thalamic state, and ultimately thalamic bursting, may play a key role in shaping more complex stimulus-evoked dynamics in the thalamocortical pathway. NEW & NOTEWORTHY: For the first time, we have been able to utilize optogenetic modulation of thalamic firing modes combined with optical imaging of cortex in the rat vibrissa system to directly test the role of thalamic state in shaping cortical response properties.

Milliseconds of Sensory Input Abruptly Modulate the Dynamics of Cortical States for Seconds.

Spontaneous internal activity plays a major role in higher brain functions. The question of how it modulates sensory evoked activity and behavior has been explored in anesthetized rodents, cats, monkeys and in behaving human subjects. However, the complementary question of how a brief sensory input modulates the internally generated activity in vivo remains unresolved, and high-resolution mapping of these bidirectional interactions was never performed. Integrating complementary methodologies, at population and single cells levels, we explored this question. Voltage-sensitive dye imaging of population activity in anesthetized rats' somatosensory cortex revealed that spontaneous up-states were largely diminished for ~2 s, even after a single weak whisker deflection. This effect was maximal at the stimulated barrel but spread across several cortical areas. A higher velocity whisker deflection evoked activity at ~15Hz. Two-photon calcium imaging activity and cell-attached recordings confirmed the VSD results and revealed that for several seconds most single cells decreased their firing, but a small number increased firing. Comparing single deflection with long train stimulation, we found a dominant effect of the first population spike. We suggest that, at the onset of a sensory input, some internal messages are silenced to prevent overloading of the processing of relevant incoming sensory information.