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Cholesterol Functionalization of Gold Nanoparticles Enhances Photo-Activation of Neural Activity
摘要： Gold nanoparticles (AuNPs) attached to the extracellular leaflet of the plasma membrane of neurons can enable the generation of action potentials (APs) in response to brief pulses of light. Recently described techniques to stably bind AuNP bioconjugates directly to membrane proteins (ion channels) in neurons enable robust AP generation mediated by the photoexcited conjugate. However, a strategy that binds the AuNP to the plasma membrane in a non-protein-specific manner could represent a simple, single-step means of establishing light-responsiveness in multiple types of excitable neurons contained in the same tissue. Based on the ability of cholesterol to insert into the plasma membrane, here we test whether AuNP functionalization with linear dihydrolipoic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP generation in neurons. Dorsal root ganglion (DRG) neurons of rat were labelled with 20 nm diameter spherical AuNP-PEG-Chol conjugates wherein ~30% of the surface ligands (DHLA-PEG-COOH) were conjugated to PEG-Chol. Voltage recordings under current-clamp conditions showed that DRG neurons labeled in this manner exhibited a capacity for AP generation in response to microsecond and millisecond pulses of 532 nm light, a property attributable to the close tethering of AuNP-PEG-Chol conjugates to the plasma membrane facilitated by the cholesterol moiety. Light-induced AP and subthreshold depolarizing responses of the DRG neurons were similar to those previously described for AuNP conjugates targeted to channel proteins using large, multicomponent immunoconjugates. This likely reflected the AuNP-PEG-Chol’s ability, upon plasmonic light absorption and resultant slight and rapid heating of the plasma membrane, to induce a concomitant transmembrane depolarizing capacitive current. Notably, AuNP-PEG-Chol delivered to DRG neurons by inclusion in the buffer contained in the recording pipette/electrode enabled similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol bound to the inner (cytofacial) leaflet of the plasma membrane. Our results demonstrate the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct responsiveness to light in neurons. Further, this strategy represents a general approach for establishing excitable cell photosensitivity that could be of substantial advantage for exploring a given tissue’s suitability for AuNP-mediated photo-control of neural activity.
关键词： nanoparticle functionalization,cholesterol,action potential,neural photo-activation,optocapacitance,gold nanoparticles,photosensitivity,dorsal root ganglion cell
更新于2025-09-23 15:23:52
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Cardiac Electrophysiological Effects of Light-Activated Chloride Channels
摘要： During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl?-driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.
关键词： heart,action potential,zebrafish,optogenetics,GtACR1,natural anion channelrhodopsin
更新于2025-09-23 15:22:29
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Optical Recording of Action Potential Initiation and Propagation in Mouse Skeletal Muscle Fibers
摘要： Skeletal muscle ?bers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[b[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-?uo-4, a low-af?nity intracellular Ca2tindicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric ?eld that initiates an AP at either end of the ?ber. Using enzymatically dissociated ?exor digitorum brevis (FDB) ?bers and high-speed line scans, we determine the conduction velocity to be (cid:2)0.4 m/s. We applied these methodologies to FDB ?bers under elevated extracellular potassium conditions and con?rmed that the conduction velocity is signi?cantly reduced in elevated [Kt]o. Because our recorded velocities for FDB ?bers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB ?bers to that of extensor digitorum longus (EDL) ?bers and measured a signi?cantly faster velocity in EDL ?bers than FDB ?bers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Nat channels in EDL than in FDB ?bers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle ?ber to predict passive polarization of the ?ber by the ?eld stimuli. Our predictions from the model ?ber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[b[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-?uo-4 to noninvasively measure AP initiation and conduction.
关键词： potentiometric dye,action potential,Nat channels,skeletal muscle,Ca2t indicator,conduction velocity,electric ?eld stimulation
更新于2025-09-23 15:21:01
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Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients
摘要： Although biomimetic stimuli, as microgroove-induced alignment (μ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined μ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of the TGF-β signaling was downregulated; the TGF-β receptor agonist and antagonist TGF-β1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.
关键词： triiodothyronine,calcium handling,electrical conditioning,tissue engineering,maturation,action potential
更新于2025-09-23 15:19:57