Developmental changes in the electrophysiological properties of neonatal rat oculomotor neurons studied in vitro

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-μm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.

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Optogenetic currents in myofibroblasts acutely alter electrophysiology and conduction of co-cultured cardiomyocytes

10.1101/2020.06.02.124529 ◽

2020 ◽

Cited By ~ 1

Author(s):

Geran Kostecki ◽

Yu Shi ◽

Christopher Chen ◽

Daniel H. Reich ◽

Emilia Entcheva ◽

...

Keyword(s):

Cardiac Tissue ◽

Cardiac Injury ◽

Neonatal Rat ◽

Culture Studies ◽

Electrophysiological Properties ◽

Inward Currents ◽

Life Threatening ◽

Cultured Cardiomyocytes ◽

Cardiac Myofibroblasts

AbstractInteractions between cardiac myofibroblasts and myocytes may slow conduction after cardiac injury, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the mechanism(s) remain debatable. In this study, primary neonatal rat cardiac myofibroblasts were transduced with the light-activated ion channel Channelrhodopsin-2, which allowed acute and selective modulation of myofibroblast currents in co-cultures with cardiomyocytes. Optical mapping revealed that myofibroblast-specific optogenetically induced inward currents decreased conduction velocity in the co-cultures by 27±6% (baseline = 17.7±5.3 cm/s), and shortened the cardiac action potential duration by 14±7% (baseline = 161±11 ms) when 0.017 mW/mm2 light was applied. When light irradiance was increased to 0.057 mW/mm2, the myofibroblast currents led to spontaneous beating in 6/7 co-cultures. Experiments showed that optogenetic perturbation did not lead to changes in myofibroblast strain and force generation, suggesting purely electrical effects in this model. In silico modeling of optogenetically modified myofibroblast-cardiomyocyte co-cultures largely reproduced these results and enabled a comprehensive study of relevant parameters. These results clearly demonstrate that myofibroblasts are sufficiently electrically connected to cardiomyocytes to effectively alter macroscopic electrophysiological properties in this model of cardiac tissue.

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Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems

AJP Cell Physiology ◽

10.1152/ajpcell.00306.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. C1256-C1267 ◽

Cited By ~ 4

Author(s):

Simon P. Wells ◽

Helen M. Waddell ◽

Choon Boon Sim ◽

Shiang Y. Lim ◽

Gabriel B. Bernasochi ◽

...

Keyword(s):

Conduction Velocity ◽

High Throughput ◽

Microelectrode Array ◽

Ventricular Myocytes ◽

Field Potential ◽

Induced Pluripotent Stem Cell ◽

Neonatal Rat ◽

Functional Screening ◽

Electrophysiological Properties

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

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Electrophysiological properties of rat lateral parabrachial neurons in vitro

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1999.276.3.r696 ◽

1999 ◽

Vol 276 (3) ◽

pp. R696-R706 ◽

Cited By ~ 1

Author(s):

Linda F. Hayward ◽

Robert B. Felder

Keyword(s):

Spatial Clustering ◽

Distinct Type ◽

Neonatal Rat ◽

Intrinsic Properties ◽

Adult Rats ◽

Membrane Hyperpolarization ◽

Electrophysiological Properties ◽

Lateral Parabrachial Nucleus ◽

A Current

Anatomical studies have demonstrated that the lateral parabrachial nucleus (LPBN) is composed of at least seven separate subnuclei distinguished by cell morphology, spatial clustering, and afferent and efferent connectivity. We hypothesized that neurons within the subnuclear clusters of the LPBN might have distinct electrophysiological properties that correlate with cellular morphology. An in vitro slice preparation was used to intracellularly record the intrinsic properties of 64 neurons located within the external lateral (EL) and central lateral (CL) subnuclei of the LPBN in adult rats. Analysis of intrinsic properties revealed that neurons in the EL subnucleus had significantly wider action potentials and on the average demonstrated more spike frequency adaptation during 2 s of depolarization compared with CL neurons. The majority of both EL and CL area neurons expressed delayed excitation (DE) after membrane hyperpolarization. DE was eliminated with the A-current blocker 4-aminopyridine (1.5–5 mM). Postinhibitory rebound was also observed in a subpopulation of EL and CL neurons. Morphological analysis of 11 LPBN neurons, which were electrophysiologically characterized and filled with 2% biocytin, failed to demonstrate an association between morphology and the electrophysiological profiles of LPBN neurons. The lack of distinct “type” of neuron within a single subnucleus of the LPBN is in agreement with recent findings reported from the neonatal rat.

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Electrophysiological properties of neonatal rat motoneurones studied in vitro.

The Journal of Physiology ◽

10.1113/jphysiol.1986.sp015956 ◽

1986 ◽

Vol 370 (1) ◽

pp. 651-678 ◽

Cited By ~ 169

Author(s):

B P Fulton ◽

K Walton

Keyword(s):

Neonatal Rat ◽

Electrophysiological Properties

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Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.2.h433 ◽

1999 ◽

Vol 277 (2) ◽

pp. H433-H444 ◽

Cited By ~ 98

Author(s):

N. Bursac ◽

M. Papadaki ◽

R. J. Cohen ◽

F. J. Schoen ◽

S. R. Eisenberg ◽

...

Keyword(s):

Cardiac Muscle ◽

Cardiac Myocytes ◽

In Vitro Model ◽

Neonatal Rat ◽

Electrophysiological Properties ◽

Impulse Propagation ◽

Ventricular Cells ◽

Electrophysiological Studies ◽

Vitro Model

The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50–70 μm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

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Developmental Changes in the Electrophysiological Properties of Brain Stem Trigeminal Neurons During Pattern (Barrelette) Formation

Journal of Neurophysiology ◽

10.1152/jn.1998.79.3.1295 ◽

1998 ◽

Vol 79 (3) ◽

pp. 1295-1306 ◽

Cited By ~ 16

Author(s):

William Guido ◽

Emine Günhan-Agar ◽

Reha S. Erzurumlu

Keyword(s):

Pattern Formation ◽

Brain Stem ◽

Current Injection ◽

Developmental Changes ◽

Postnatal Life ◽

Action Potential Firing ◽

Electrophysiological Properties ◽

Trigeminal Neurons ◽

The Brain

Guido, William, Emine Günhan-Agar, and Reha S. Erzurumlu. Developmental changes in the electrophysiological properties of brain stem trigeminal neurons during pattern (barrelette) formation. J. Neurophysiol. 79: 1295–1306, 1998. In the brain stem trigeminal nuclei of rodents there is a patterned representation of whiskers and sinus hairs. The subnucleus interpolaris (SPI) contains the largest and the most conspicuous whisker patterns (barrelettes). Although neural activity plays a role in pattern formation, little is known about the electrophysiological properties of developing barrelette neurons. Here we examined the functional state of early postnatal SPI neurons during and after the consolidation of patterns by using in vitro intracellular recording techniques. After the consolidation of barrelettes [≥ postnatal day (P)4], responses to intracellular current injection consistently reflected the activation of a number voltage-dependent conductances. Most notable was a mixed cation conductance ( I H) that prevented strong hyperpolarization and a large low-threshold Ca2+ conductance, which led to Ca2+ spikes and burst firing. At the oldest ages tested (P11–P14) some cells also exhibited an outward K+ conductance ( I A), which led to significant delays in action-potential firing. Between P0–3, a time when the formation of barrelettes in the brain stem is still susceptible to damage of the sensory periphery, cells responded linearly to intracellular current injection, indicating they either lacked such voltage-gated properties or weakly expressed them. At all ages tested (P0–14), SPI cells were capable of generating trains of action potentials in response to intracellular injection of depolarizing current pulses. However, during the first few days of postnatal life, spikes were shorter and longer. Additionally, spike trains rose more linearly with stimulus intensity and showed frequency accommodation at early ages. Taken together, these results indicate that the electrophysiological properties of SPI neurons change markedly during the period of barrelette consolidation. Moreover, the properties of developing SPI neurons may play a significant role in pattern formation by minimizing signal distortion and ensuring that excitatory responses from sensory periphery are accurately received and transmitted according to stimulus strength.

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Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079590 ◽

1977 ◽

Vol 35 ◽

pp. 430-431

Author(s):

L.S. Cutler

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Submandibular Gland ◽

Neonatal Rat ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Parotid Glands ◽

Adrenergic Agonist ◽

Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

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