Evaluation of Excitation Propagation in the Rabbit Heart: Optical Mapping and Transmural Microelectrode Recordings

Background

Because of the optical features of heart tissue, optical and electrical action potentials are only moderately associated, especially when near-infrared dyes are used in optical mapping (OM) studies.

Objective

By simultaneously recording transmural electrical action potentials (APs) and optical action potentials (OAPs), we aimed to evaluate the contributions of both electrical and optical influences to the shape of the OAP upstroke.

Methods and Results

A standard glass microelectrode and OM, using an near-infrared fluorescent dye (di-4-ANBDQBS), were used to simultaneously record transmural APs and OAPs in a Langendorff-perfused rabbit heart during atrial, endocardial, and epicardial pacing. The actual profile of the transmural AP upstroke across the LV wall, together with the OAP upstroke, allowed for calculations of the probing-depth constant (k ~2.1 mm, n = 24) of the fluorescence measurements. In addition, the transmural AP recordings aided the quantitative evaluation of the influences of depth-weighted and lateral-scattering components on the OAP upstroke. These components correspond to the components of the propagating electrical wave that are transmural and parallel to the epicardium. The calculated mean values for the depth-weighted and lateral-scattering components, whose sum comprises the OAP upstroke, were (in ms) 10.18 ± 0.62 and 0.0 ± 0.56 for atrial stimulation, 9.37 ± 1.12 and 3.01 ± 1.30 for endocardial stimulation, and 6.09 ± 0.79 and 8.16 ± 0.98 for epicardial stimulation; (n = 8 for each). For this dye, 90% of the collected fluorescence originated up to 4.83 ± 0.18 mm (n = 24) from the epicardium.

Conclusions

The co-registration of OM and transmural microelectrode APs enabled the probing depth of fluorescence measurements to be calculated and the OAP upstroke to be divided into two components (depth-weighted and lateral-scattering), and it also allowed the relative strengths of their effects on the shape of the OAP upstroke to be evaluated.     

Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish

Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development.     

Left Ventricular Stresses in the Intact Human Heart

A set of stress differential equations of equilibrium is presented for a thick prolate spheroid which is the assumed shape for the left ventricle. An analysis for the stresses in the ventricular wall indicates that maximum stresses occur at the inner layers and decrease to a minimum at the epicardial surface, a result that is partially validated by experiment. Simple expressions are available for the evaluation of maximum stresses which occur at the equator and are suitable for small laboratory-oriented digital computers employed in the clinical evaluation of patient status. The surprising result is that Laplace's law yields practical values for mean stresses in thick-walled ventricles.     

Functional Morphology of the Heart in Fishes

The systemic heart of fishes consists of four chambers in series,the sinus venosus, atrium, ventricle, and conus or bulbus. Valvesbetween the chambers and contraction of all chambers exceptthe bulbus maintain a unidirectional blood flow through theheart. The heart is composed of typical vertebrate cardiac muscle,although there may be minor differences in the distributionof spontaneously active cells, the rate and nature of spreadof excitatory waves, and the characteristics of resting andaction potentials between different fish and other vertebrates.Cholinergic fibers innervate the heart, except in hagfish whichhave aneural hearts. Fish hearts lack sympathetic innervation.The level of vagal tone varies considerably, and is affectedby many factors. In some fish the heart is essentially aneural(without vagal tone) during exercise and may resemble an isolatedmammalian ventricle with increased venous return causing increasedcardiac output. There are many mechanisms that could increasevenous return in exercising fish. rß-adrenergic receptorshave been located on the hearts of some fish, and changing levelsof catecholamines may play a role in regulating cardiac activity.Changes in cardiac output in fish are normally associated withlarge changes in stroke volume and small cha-nges in heart rate.     

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals¹. Though being a crucial part of the visual processing by shaping the retinal output², most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue^3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage⁵. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments⁶.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.     

Preparation and Characterization of Intact Globular Cell Wall Peptidoglycan from Micrococcus lysodeikticus

The intact globular cell wall peptidoglycan was prepared from Micrococcus lysodeikticus without any mechanical disruption. The purification procedure consists mainly of proteolytic digestion and extractions with hot 5% TCA and with 0.01 N NaOH. The purified preparation showed an amphoteric, heteroporous three-dimensional network structure of the peptidoglycan, which amounted to 77% of the dry weight of the preparation, retaining the chemical and morphological integrity as judged by an electron microscopic observation and chemical analyses.     

Coupling of Mitochondrial Fatty Acid Uptake to Oxidative Flux in the Intact Heart

The coordination of long chain fatty acid (LCFA) transport across the mitochondrial membrane (V_PAL) with subsequent oxidation rate through β-oxidation and the tricarboxylic acid (TCA) cycle (V_tca) has been difficult to characterize in the intact heart. Kinetic analysis of dynamic ¹³C-NMR distinguished these flux rates in isolated rabbit hearts. Hearts were perfused in a 9.4 T magnet with either 0.5 mM [2,4,6,8,10,12,14,16-¹³C₈] palmitate (n = 4), or 0.5 mM ¹³C-labeled palmitate plus 0.08 mM unlabeled butyrate (n = 4). Butyrate is a short chain fatty acid (SCFA) that bypasses the LCFA transporters of mitochondria. In hearts oxidizing palmitate alone, the ratio of V_TCA to V_PAL was 8:1. This is consistent with one molecule of palmitate yielding eight molecules of acetyl-CoA for the subsequent oxidation through the TCA cycle. Addition of butyrate elevated this ratio; V_TCA/V_PAL = 12:1 due to an SCFA-induced increase in V_TCA of 43% (p < 0.05). However, SCFA oxidation did not significantly reduce palmitate transport into the mitochondria: V_PAL = 1.0 ± 0.2 μmol/min/g dw with palmitate alone versus 0.9 ± 0.1 with palmitate plus butyrate. Thus, the products of β-oxidation are preferentially channeled to the TCA cycle, away from mitochondrial efflux via carnitine acetyltransferase.     

Cortical Spreading Depolarization (CSD) Recorded from Intact Skin,from Surface of Dura Mater or Cortex: Comparison with Intracortical Recordings in the Neocortex of Adult Rats

In cerebral cortex of anesthetized rats single waves of spreading depolarization (CSD) were elicited by needle prick. CSD-related changes of DC (direct current) potentials were either recorded from the intact skin or together with concomitant changes of potassium concentration with K⁺-selective microelectrodes simultaneously at the surface of the dura mater or of the cortex ([K⁺]_s) and in the extracellular space at a cortical depth of 1200 µm. At the intact skin CSD-related DC-shifts had amplitudes of less than 1 mV and had only in a minority of cases the typical CSD-like shape. In the majority these DC-shifts rose and recovered very slowly and were difficult to identify without further indicators. At dura surface CSD-related DC shifts were significantly smaller and rose and recovered slower than intracortically recorded CSD. Concomitant increases in [K⁺]_s were delayed and reached maximal values of about 5 mM from a baseline of 3 mM. They rose and recovered slower than simultaneously recorded intracortical changes in extracellular potassium concentration ([K⁺]_e) that were up to 65 mM. The results suggest that extracellular potassium during CSD is diffusing through the subarachnoid space and across the dura mater. In a few cases CSD was either absent at the dura or at a depth of 1200 µm. Even full blown CSDs in this cortical depth could remain without concomitant deflections at the dura. Our data confirmed in principle the possibility of non-invasive recordings of CSD-related DC-shifts. For a use in clinical routine sensitivity and specificity will have to be improved.

     

Validity of (Ultra-)Short Recordings for Heart Rate Variability Measurements

Objectives

In order to investigate the applicability of routine 10s electrocardiogram (ECG) recordings for time-domain heart rate variability (HRV) calculation we explored to what extent these (ultra-)short recordings capture the “actual” HRV.

Methods

The standard deviation of normal-to-normal intervals (SDNN) and the root mean square of successive differences (RMSSD) were measured in 3,387 adults. SDNN and RMSSD were assessed from (ultra)short recordings of 10s(3x), 30s, and 120s and compared to 240s–300s (gold standard) measurements. Pearson’s correlation coefficients (r), Bland-Altman 95% limits of agreement and Cohen’s d statistics were used as agreement analysis techniques.

Results

Agreement between the separate 10s recordings and the 240s-300s recording was already substantial (r = 0.758–0.764/Bias = 0.398–0.416/d = 0.855–0.894 for SDNN; r = 0.853–0.862/Bias = 0.079–0.096/d = 0.150–0.171 for RMSSD), and improved further when three 10s periods were averaged (r = 0.863/Bias = 0.406/d = 0.874 for SDNN; r = 0.941/Bias = 0.088/d = 0.167 for RMSSD). Agreement increased with recording length and reached near perfect agreement at 120s (r = 0.956/Bias = 0.064/d = 0.137 for SDNN; r = 0.986/Bias = 0.014/d = 0.027 for RMSSD). For all recording lengths and agreement measures, RMSSD outperformed SDNN.

Conclusions

Our results confirm that it is unnecessary to use recordings longer than 120s to obtain accurate measures of RMSSD and SDNN in the time domain. Even a single 10s (standard ECG) recording yields a valid RMSSD measurement, although an average over multiple 10s ECGs is preferable. For SDNN we would recommend either 30s or multiple 10s ECGs. Future research projects using time-domain HRV parameters, e.g. genetic epidemiological studies, could calculate HRV from (ultra-)short ECGs enabling such projects to be performed at a large scale.     

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact,Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.Download video file.^{(145M, mp4)}     

Vectorcardiographic Abnormalities Simulating Myocardial Infarction in the Normal Heart

During a study of the vectorcardiogram in the normal young heart, four individuals were found in whom the QRS loops, surprisingly, fulfilled the usual criteria for the diagnosis of myocardial infarction. These vectorcardiograms are described in detail. Two of the four electrocardiograms are within normal limits. These abnormal vectorcardiographic patterns are probably due to a congenital variant in left ventricular conduction. This rare condition is of clinical significance because it may result in diagnostic confusion in older patients.     

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.     

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.