Electrophysiological Membrane Research Articles

Integrative Modeling of Functional Hyperemia: From Ion Channel Activity to Functional Imaging

Neuronal activity signals local changes in functional hyperemia which match metabolic demands for oxygen (O2) -and other nutrients, referred to as Neurovascular Coupling (NVC). NVC is essential for proper functioning of the brain and is impaired in various clinical conditions including Alzheimer's disease, stroke and aging (Phillips, et al., JCBFM, 2016). The emerging paradigm is that neurons and astrocytes release vasoactive mediators in the perivascular space of penetrating arterioles to increase their diameter and blood supply. Recent evidence supports the concept that brain capillaries can also act as a neuronal activity-sensing network, and initiate electrical signal that propagate upstream to cause dilation and increase local blood flow (Longden, et al., Nature, 2017). How signaling of neuronal activity to the capillaries intersects with arteriole-level communication is completely unknown. In this study, we propose an integrative modeling approach to model microcirculatory responses to NVC mediators and their effect on the regulation of blood flow, tissue perfusion, and oxygenation. Single cell models of endothelial cells (EC) and smooth muscle cells (SMC) describe membrane electrophysiology and Ca2+ dynamics. Cells are coupled through gap junctions to form branched capillary networks connected to parenchymal arterioles to examine capillary to arteriole communication. Hemodynamic responses are governed by conservation of flow, Fåhræus–Lindqvist effect, and the phase separation effect (Pries, Secomb, AJPHAP, 2005). Oxygen simulations provide PO2 within vessels and in the tissue. Simulations are extended to a macroscale level by incorporating reconstructed brain vascular networks from (Blinder, et al., Nature, 2013). The model predicts changes in tissue perfusion and oxygen distribution in response to neuronal activity. The model accounts for dynamic regulation of PA diameters by NVC mediators and propagating electrical signals from connected capillary beds. Simulations suggest an important role of capillary-level NVC in regulating functional hyperemia. The theoretical framework presented allows for testing proposed NVC mechanisms and assisting in the interpretation of macroscale functional imaging responses in health and in disease. Support or Funding Information This work was supported by NIH grant 1R15HL121778-01A1 A, B) Cell models for capillary EC, parenchymal arteriole EC and SMC simulate electrophysiology and Ca2+ dynamics in response to mechanical stimuli or NVC mediators. C) Multicellular models of a Capillary and a PA are constructed. D) Capillaries are coupled to a PA to form a local vascular network and detailed simulations of blood flow and O2 distribution are performed. E) Predicted changes in Oxy/Deoxy Hb distribution are translated to responses in functional imaging scans such as BOLD fMRI. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid determinants of endocytosis and exocytosis in budding yeast

Cells maintain physicochemical characteristics of membranes in order to allow for proper function of membrane-associated cellular processes, such as endocytosis and exocytosis. To investigate the interplay between membrane properties and biological processes, we applied lipid engineering approaches that allowed for systematic manipulation of fatty acid unsaturation and sterol biosynthesis, the main regulators of membrane fluidity. In combination with electrophysiological membrane capacitance measurements, we were able to study the dependence of the endo- and exocytic activity of Saccharomyces cerevisiae on membrane lipid composition in vivo. We found that a strong decrease in the cell's total ergosterol content leads to a severely reduced frequency of vesicle fission (endocytosis), whereas the exocytic activity remained largely unaffected. In contrast, increased lipid saturation lowered both endocytic and the exocytic activity, with the former being more severely affected. We were able to correlate the decreased ratio of endocytic/exocytic frequencies (fendo/fexo) upon lipid perturbation with the growth of yeast protoplasts, which is based on a surface enlargement resulting from a net excess of exocytic over endocytic flux. Experiments using clathrin-deficient mutants confirm a correlation between reduced endocytic activity and increased size of intact walled cells, as well as accelerated protoplast growth. These data show that lipid composition is intimately tied to membrane trafficking in yeast cells and suggest that endocytosis is particularly dependent on the lipid-defined properties of cell membrane.

Effects of Varying Transverse and Axial Tubules in a Three-Dimensional Model of Calcium Signaling in the Human Atrial Myocyte

T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in ventricular myocytes. Atrial myocytes are generally thought to possess sparse irregular transverse-tubular (TT) components, as opposed to the highly dense and regular ventricular TT system. Axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum that include ryanodine receptor (RyR) clusters, characterized by rapid activation of Ca2+-induced Ca2+ release, have also been identified in atrial myocytes. AT and TT remodeling and changes in distribution, composition, and phosphorylation status of Ca2+ release units are thought to underlie Ca2+ abnormalities in atrial fibrillation (AF), the most common cardiac arrhythmia. Here, we performed a computational analysis to investigate how changes in the tubular network affect human atrial electrophysiology. We modified our well-established three-dimensional model of Ca2+ signaling in the rabbit ventricular myocyte to develop an analogous model in the human atrium. We also coupled the Ca2+ signaling model to our well-established model of membrane electrophysiology in the human atrial myocyte. We systematically varied TT and AT density and RyR distribution and assessed the effect on Ca2+ spark and wave properties. When TT density is low, as shown in isolated atrial myocytes, the model recapitulates the typical U-shaped Ca2+ wave seen experimentally in transverse confocal line-scan images. Introduction of ATs resulted in W-shaped Ca2+ waves, reflecting more synchronous Ca2+ release. The frequency and amplitude of Ca2+ release events were affected by both density of TT and AT, as well as RyR distribution. Our newly developed three-dimensional model of the human atrial myocyte will be employed to investigate whether and how AF-induced cellular electrical and structural remodeling effects collectively contribute to the membrane potential and Ca2+ abnormalities seen in AF.

A Mathematical Model of Plasma Membrane Electrophysiology of a Brain Capillary Pericyte: Investigating Pericyte Contribution to the Electrical Properties of the Capillary Network

IntroductionPericyte cells (PCs) are specialized cells found primarily on the abluminal surface of capillary blood vessels alongside endothelial cells (ECs). They play a role in angiogenesis, formation of the blood‐brain barrier, as well as in the regulation of blood flow. Due to their contractile nature, PCs can modulate capillary diameter and perfusion, however, little is known regarding their contribution to the electrical properties of the capillary network. Furthermore, recent studies show that capillaries can sense neural activity in the brain and transmit electrical signals to upstream feeding arterioles and arteries to match blood supply to local demand. Although passive electrotonic spread underlies signal propagation along the endothelium in larger vessels, evidence for amplification of transmitted signals and preferential upstream propagation have been presented in vessels as branching order increases. We use mathematical modeling to test under what conditions a PC can affect the electrical properties of the capillary network and whether it can act as a sink or an amplifier of conducted hyperpolarization.MethodsA PC and an EC model incorporate the dynamic behavior of known plasma membrane currents, release and uptake of Ca2+ by intracellular stores and dynamic tracking of ionic (Ca2+, K+, Na+, and Cl−) concentrations in the cytosol (Fig. 1). The PC is electrically coupled to capillary ECs through gap junctions and a multicellular model of a capillary network is created. Electrical signal attenuation is examined following hyperpolarizing stimuli at capillary ends in the presence as well as absence of PCs at network bifurcations.ResultsCell models predict physiological resting membrane potential and ionic concentrations. Model predictions were compared against experimental responses to agonist stimulation and K+ challenge for validation. Simulations show that a sufficient inwardly rectifying K+, Kir, channel current density may allow PCs to amplify conducted hyperpolarization. Increased coupling between a PC with the parent relative to the daughter capillaries at a bifurcation may allow preferential conduction of an electrical signal upstream the vascular network.ConclusionA detailed model of a brain capillary pericyte was developed and validated against experimental data. Model simulations suggest that with sufficient Kir density, pericytes can amplify conducted hyperpolarization and regulate the distance and direction of propagating vasodilation and thus blood flow distribution in the vascular network.Support or Funding InformationThis work was supported by the Ronald E. McNair Post‐Baccalaureate Achievement Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biophysical Characterization of Lipid Membranes: Effect of Lipid Head Groups and Tails on Synthetic and Natural Lipid Membranes

Lipid membranes play important roles in cell biology. They serve as a structure that protects cellular organelles and maintains unique cell shapes and also as a regulator that controls cellular functions. Thus, understanding the biophysical characteristics lipid membranes, e.g., fluidity and elasticity, is essential, as membrane proteins in cells often modulate these characteristics to properly control cellular activities. Here, we investigate various lipid head groups and tails by utilizing three different experimental techniques: 1) fluorescence recovery after photobleaching (FRAP), 2) black lipid membrane (BLM) electrophysiology recordings, and 3) atomic force microscopy (AFM). Our results show lipid membranes composed of either negatively charged headgroups or lactocylated headgroups have slower lateral diffusion coefficients and reduced elasticity compared to lipids with neutrally charged headgroups or zwitterionic headgroups. Addition of methyl groups in the acyl chain of the lipids decreases lateral diffusion but does not change elasticity of the membrane. Further, we study the biophysical characteristics of two natural lipid membranes extracted from E. coli and P. furiosus, which have known lipid compositions are distinct from each other, and compare our findings from synthetic lipids to understand the biophysical characteristics of natural membranes. We observed that P. furiosus lipid membranes exhibit a slower lateral diffusion and less elasticity compared with E. coli lipid membranes. From correlation among lateral diffusion, gA lifetime (local bending energy), and elasticity, we found that lipid membranes with slower lateral diffusion shows bigger local bending energy and membrane elastic modulus.

Electrophysiological and Morphological Properties of α and γ Motoneurons in the Rat Trigeminal Motor Nucleus.

The muscle contraction during voluntary movement is regulated by activities of α- and γ-motoneurons (αMNs and γMNs, respectively). The tension of jaw-closing muscles can be finely tuned over a wide range. This excellent function is likely to be achieved by the specific populations of αMNs innervating jaw-closing muscles. Indeed, we have recently demonstrated that in the rat dorsolateral trigeminal motor nucleus (dl-TMN), the size distribution of αMNs was bimodal and the population of smaller αMNs showed a size distribution similar to that of γMNs, by immunohistochemically identifying αMNs and γMNs based on the expressions of estrogen-related receptor gamma (Err3) and neuronal DNA binding protein NeuN together with ChAT. This finding suggests the presence of αMNs as small as γMNs. However, differences in the electrophysiological membrane properties between αMNs and γMNs remain unknown also in the dl-TMN. Therefore, in the present study, we studied the electrophysiological membrane properties of MNs in the dl-TMN of infant rats at postnatal days 7–12 together with their morphological properties using whole-cell current-clamp recordings followed by immunohistochemical staining with an anti-NeuN and anti-ChAT antibodies. We found that the ChAT-positive and NeuN-positive αMNs were divided into two subclasses: the first one had a larger cell body and displayed a 4-aminopyridine (4-AP)-sensitive current while the second one had a smaller cell body and displayed a less prominent 4-AP-sensitive current and a low-threshold spike, suitable for their orderly recruitment. We finally found that γMNs showing ChAT-positive and NeuN-negative immunoreactivities had smaller cell bodies and displayed an afterdepolarization mediated by flufenamate-sensitive cation current. It is suggested that these electrophysiological and morphological features of MNs in the dl-TMN are well correlated with the precise control of occlusion.

Cortical and spinal conditioned media modify the inward ion currents and excitability and promote differentiation of human striatal primordium

Human striatal precursor cells (HSPs) isolated from ganglionic eminence may differentiate in electrophysiologically functional excitable neuron-like cells and a number of endogenous molecules such as hormones, neurotransmitters or growth factors can actually regulate neuronal growing and differentiation. The purpose of this research was to assess, by electrophysiological and immunocytochemical analysis, if the type of culture medium could specifically impact on the neuronal differentiation potential of HSPs. Accordingly, HSPs were maintained in different inductive media such as cortical and spinal cord conditioned media, and we estimated the possible changes in the main ion currents, excitability and expression of neuronal markers indicative of neuronal differentiation. Our results have shown that 36 h exposure to each of the conditioned media, with their blend of autocrine and paracrine growth factors, was able to modify significantly the electrophysiological membrane properties and the functional expression of inward ionic currents in selected neuronal HSPs. Moreover, although both types of conditioned media determined neuronal maturation (increased neuritogenesis and increased expression of neuronal and striatal markers), each of them leads to the occurrence of different functional features. Particularly, the spinal medium caused a stronger depolarization of the membrane potential and significantly increased the amplitude of Na+ current as well as L- and N- type Ca2+ currents, definitely modifying their kinetics. In contrast, the cortical medium mainly caused a significant and more marked increase of the membrane conductance and time constant values. These results strongly support the plasticity of our cellular model that, although already committed towards a specific phenotype, it can be differently affected by the conditioned media, thereby resulting functionally modifiable according to environmental cues.

Differential processing in modality-specific Mauthner cell dendrites.

The present study examines dendritic integrative processes that occur in many central neurons but have been challenging to study in vivo in the vertebrate brain. The Mauthner cell of goldfish receives auditory and visual information via two separate dendrites, providing a privileged scenario for in vivo examination of dendritic integration. The results show differential attenuation properties in the Mauthner cell dendrites arising at least partly from differences in cable properties and the nonlinear behaviour of the respective dendritic membranes. In addition to distinct modality-dependent membrane specialization in neighbouringdendrites of the Mauthner cell, we report cross-modal dendritic interactions via backpropagating postsynaptic potentials. Broadly, the results of the present study provide an exceptional example for the processing power of single neurons. Animals process multimodal information for adaptive behavioural decisions. In fish, evasion of a diving bird that breaks the water surface depends on integrating visual and auditory stimuli with very different characteristics. How do neurons process such differential sensory inputs at the dendritic level? For that, we studied the Mauthner cells (M-cells) in the goldfish startle circuit, which receive visual and auditory inputs via two separate dendrites, both accessible for in vivo recordings. We investigated whether electrophysiological membrane properties and dendrite morphology, studied in vivo, play a role in selective sensory processing in the M-cell. The results obtained show that anatomical and electrophysiological differences between the dendrites combine to produce stronger attenuation of visually evoked postsynaptic potentials (PSPs) than to auditory evoked PSPs. Interestingly, our recordings showed also cross-modal dendritic interaction because auditory evoked PSPs invade the ventral dendrite (VD), as well as the opposite where visual PSPs invade the lateral dendrite (LD). However, these interactions were asymmetrical, with auditory PSPs being more prominent in the VD than visual PSPs in the LD. Modelling experiments imply that this asymmetry is caused by active conductances expressed in the proximal segments of the VD. The results obtained in the present study suggest modality-dependent membrane specialization in M-cell dendrites suited for processing stimuli of different time domains and, more broadly, provide a compelling example of information processing in single neurons.

Rhythmic potassium transport regulates the circadian clock in human red blood cells

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Anti-epileptic effect of ifenprodil on neocortical pyramidal neurons in patients with malformations of cortical development.

Ifenprodil has been demonstrated to reduce spontaneous action potentials observed by local field potential in animal models. To investigate whether ifenprodil exerts an anti-epileptic effect on neuronal levels in humans, whole-cell patch clamp recordings were used to study the electrophysiological membrane properties of neocortical pyramidal neurons in human brain tissues. Electrophysiological membrane properties and spontaneous spikes of neocortical pyramidal neurons were investigated by using whole-cell patch clamp recordings, prior to and following the application of ifenprodil. In the present study, ifenprodil significantly decreased the membrane input resistance (P<0.01), membrane time constant (P<0.01), action potential amplitude (P<0.01), action potential rising rate (P<0.05) and falling rate (P<0.05) on neocortical pyramidal neurons in patients with epilepsy caused by malformations of cortical development (MCD). These results suggested that ifenprodil decreased neuronal excitability of neocortical pyramidal neurons in patients with epilepsy and MCD and demonstrated that ifenprodil may be a potentially specific treatment for refractory epilepsy caused by MCD.

Endocannabinoid-dependent disinhibition of orexinergic neurons: Electrophysiological evidence in leptin-knockout obese mice

ObjectivesIn the ob/ob mouse model of obesity, chronic absence of leptin causes a significant increase of orexin (OX) production by hypothalamic neurons and excessive food intake. The altered OX level is linked to a dramatic increase of the inhibitory innervation of OX producing neurons (OX neurons) and the over expression of the endocannabinoid 2-arachidonoylglycerol (2-AG) by OX neurons of ob/ob mice. Little is known about the function of the excitatory synapses of OX neurons in ob/ob mice, and their modulation by 2-AG. In the present study, we fill this gap and provide the first evidence of the overall level of activation of OX neurons in the ob/ob mice. MethodsWe performed in vitro whole-cell patch-clamp recordings on OX neurons located in the perifornical area of the lateral hypothalamus in acute brain slices of wt and ob/ob mice. We identified OX neurons on the basis of their electrophysiological membrane properties, with 96% of concordance with immunohistochemisty. ResultsWe found that OX neurons of ob/ob mice are innervated by less efficient and fewer excitatory synapses than wt mice. Consequently, ob/ob OX neurons show more negative resting membrane potential and lower action potential firing frequency than wt. The bath application of the cannabinoid type-1 receptor agonist WIN55,212-2, depresses both the excitatory and the inhibitory synapses in ob/ob animals, but only the excitatory synapses in wt animals. Finally, the physiologic release of 2-AG induces a prevalent depression of inhibition (disinhibition) of OX neurons in ob/ob animals but not in wt. ConclusionsIn ob/ob mice, chronic absence of leptin induces a 2-AG mediated functional disinhibition of OX neurons. This helps explain the increase of OX production and, consequently, the excessive food intake of ob/ob mice.

Can endothelial hemoglobin-α regulate nitric oxide vasodilatory signaling?

We used mathematical modeling to investigate nitric oxide (NO)-dependent vasodilatory signaling in the arteriolar wall. Detailed continuum cellular models of calcium (Ca2+) dynamics and membrane electrophysiology in smooth muscle and endothelial cells (EC) were coupled with models of NO signaling and biotransport in an arteriole. We used this theoretical approach to examine the role of endothelial hemoglobin-α (Hbα) as a modulator of NO-mediated myoendothelial feedback, as previously suggested in Straub et al. (Nature 491: 473-477, 2012). The model considers enriched expression of inositol 1,4,5-triphosphate receptors (IP3Rs), endothelial nitric oxide synthase (eNOS) enzyme, Ca2+-activated potassium (KCa) channels and Hbα in myoendothelial projections (MPs) between the two cell layers. The model suggests that NO-mediated myoendothelial feedback is plausible if a significant percentage of eNOS is localized within or near the myoendothelial projection. Model results show that the ability of Hbα to regulate the myoendothelial feedback is conditional to its colocalization with eNOS near MPs at concentrations in the high nanomolar range (>0.2 μM or 24,000 molecules). Simulations also show that the effect of Hbα observed in in vitro experimental studies may overestimate its contribution in vivo, in the presence of blood perfusion. Thus, additional experimentation is required to quantify the presence and spatial distribution of Hbα in the EC, as well as to test that the strong effect of Hbα on NO signaling seen in vitro, translates also into a physiologically relevant response in vivo.NEW & NOTEWORTHY Mathematical modeling shows that although regulation of nitric oxide signaling by hemoglobin-α (Hbα) is plausible, it is conditional to its presence in significant concentrations colocalized with endothelial nitric oxide synthase in myoendothelial projections. Additional experimentation is required to test that the strong effect of Hbα seen in vitro translates into a physiologically relevant response in vivo.

Relationship of membrane properties, spike burst responses, laminar location, and functional class of dorsal horn neurons recorded in vitro.

The input-output and discharge properties of neurons are shaped by both passive and active electrophysiological membrane properties. Whole cell patch-clamp recordings in lamina I-III neurons in an isolated preparation of the whole spinal cord of juvenile rats with attached dorsal roots and dorsal root ganglia were used to further define which of these properties provides the most impactful classification strategy. A total of 95 neurons were recorded in segment L5 and were classified based on the responses to L4 dorsal root stimulation. The results showed that high-threshold and silent neurons had higher membrane resistance and more negative resting membrane potential than low-threshold or wide-dynamic-range neurons. Rheobase in low-threshold and wide-dynamic-range neurons was significantly lower than that of high-threshold or silent neurons. Four types of firing patterns were identified in response to depolarizing current injections. Low-threshold cells most frequently showed a phasic firing pattern characterized by a short initial burst of action potentials, single spiking or irregular firing bursts at the onset of a depolarizing pulse. High-threshold and wide-dynamic-range neurons were characterized by tonic firing with trains of spikes occurring at regular intervals throughout the current pulse. The majority of silent neurons displayed a delayed onset of firing in response to current injection. These results indicate that the passive membrane properties of spinal neurons are tuned to optimize the responses to particular subsets of afferent stimuli.

Automated detection and analysis of depolarization events in human cardiomyocytes using MaDEC

Optical imaging-based methods for assessing the membrane electrophysiology of in vitro human cardiac cells allow for non-invasive temporal assessment of the effect of drugs and other stimuli. Automated methods for detecting and analyzing the depolarization events (DEs) in image-based data allow quantitative assessment of these different treatments. In this study, we use 2-photon microscopy of fluorescent voltage-sensitive dyes (VSDs) to capture the membrane voltage of actively beating human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). We built a custom and freely available Matlab software, called MaDEC, to detect, quantify, and compare DEs of hiPS-CMs treated with the β-adrenergic drugs, propranolol and isoproterenol. The efficacy of our software is quantified by comparing detection results against manual DE detection by expert analysts, and comparing DE analysis results to known drug-induced electrophysiological effects. The software accurately detected DEs with true positive rates of 98–100% and false positive rates of 1–2%, at signal-to-noise ratios (SNRs) of 5 and above. The MaDEC software was also able to distinguish control DEs from drug-treated DEs both immediately as well as 10min after drug administration.

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology.

Lysosomal calcium (Ca2+) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca2+ signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2’s true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca2+ transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K+ as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation.

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

TRPV1 expression level in isolectin B₄-positive neurons contributes to mouse strain difference in cutaneous thermal nociceptive sensitivity.

Differential thermal nociception across inbred mouse strains has genetic determinants. Thermal nociception is largely attributed to the heat/capsaicin receptor transient receptor potential vanilloid 1 (TRPV1); however, the contribution of this channel to the genetics of thermal nociception has not been revealed. In this study we compared TRPV1 expression levels and electrophysiological properties in primary sensory neurons and thermal nociceptive behaviors between two (C57BL/6 and BALB/c) inbred mouse strains. Using immunofluorescence and patch-clamp physiology methods, we demonstrated that TRPV1 expression was significantly higher in isolectin B4 (IB4)-positive trigeminal sensory neurons of C57BL/6 relative to BALB/c; the expression in IB4-negative neurons was similar between the strains. Furthermore, using electrophysiological cell classification (current signature method), we showed differences between the two strains in capsaicin sensitivity in IB4-positive neuronal cell types 2 and 13, which were previously reported as skin nociceptors. Otherwise electrophysiological membrane properties of the classified cell types were similar in the two mouse strains. In publicly available nocifensive behavior data and our own behavior data from the using the two mouse strains, C57BL/6 exhibited higher sensitivity to heat stimulation than BALB/c, independent of sex and anatomical location of thermal testing (the tail, hind paw, and whisker pad). The TRPV1-selective antagonist JNJ-17203212 inhibited thermal nociception in both strains; however, removing IB4-positive trigeminal sensory neurons with IB4-conjugated saporin inhibited thermal nociception on the whisker pad in C57BL/6 but not in BALB/c. These results suggest that TRPV1 expression levels in IB4-positive type 2 and 13 neurons contributed to differential thermal nociception in skin of C57BL/6 compared with BALB/c.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

Integration of modeling with experimental and clinical findings synthesizes and refines the central role of inositol 1,4,5-trisphosphate receptor 1 in spinocerebellar ataxia.

A suite of models was developed to study the role of inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in spinocerebellar ataxias (SCAs). Several SCAs are linked to reduced abundance of IP3R1 or to supranormal sensitivity of the receptor to activation by its ligand inositol 1,4,5-trisphosphate (IP3). Detailed multidimensional models have been created to simulate biochemical calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons. In these models, IP3R1-mediated calcium release is allowed to interact with ion channel response on the cell membrane. Experimental findings in mice and clinical observations in humans provide data input for the models. The SCA modeling suite helps interpret experimental results and provides suggestions to guide experiments. The models predict IP3R1 supersensitivity in SCA1 and compensatory mechanisms in SCA1, SCA2, and SCA3. Simulations explain the impact of calcium buffer proteins. Results show that IP3R1-mediated calcium release activates voltage-gated calcium-activated potassium channels in the plasma membrane. The SCA modeling suite unifies observations from experiments in a number of SCAs. The cadre of simulations demonstrates the central role of IP3R1.

Multiscale Model of Dynamic Neuromodulation Integrating Neuropeptide-Induced Signaling Pathway Activity with Membrane Electrophysiology

We developed a multiscale model to bridge neuropeptide receptor-activated signaling pathway activity with membrane electrophysiology. Typically, the neuromodulation of biochemical signaling and biophysics have been investigated separately in modeling studies. We studied the effects of Angiotensin II (AngII) on neuronal excitability changes mediated by signaling dynamics and downstream phosphorylation of ion channels. Experiments have shown that AngII binding to the AngII receptor type-1 elicits baseline-dependent regulation of cytosolic Ca2+ signaling. Our model simulations revealed a baseline Ca2+-dependent response to AngII receptor type-1 activation by AngII. Consistent with experimental observations, AngII evoked a rise in Ca2+ when starting at a low baseline Ca2+ level, and a decrease in Ca2+ when starting at a higher baseline. Our analysis predicted that the kinetics of Ca2+ transport into the endoplasmic reticulum play a critical role in shaping the Ca2+ response. The Ca2+ baseline also influenced the AngII-induced excitability changes such that lower Ca2+ levels were associated with a larger firing rate increase. We examined the relative contributions of signaling kinases protein kinase C and Ca2+/Calmodulin-dependent protein kinase II to AngII-mediated excitability changes by simulating activity blockade individually and in combination. We found that protein kinase C selectively controlled firing rate adaptation whereas Ca2+/Calmodulin-dependent protein kinase II induced a delayed effect on the firing rate increase. We tested whether signaling kinetics were necessary for the dynamic effects of AngII on excitability by simulating three scenarios of AngII-mediated KDR channel phosphorylation: (1), an increased steady state; (2), a step-change increase; and (3), dynamic modulation. Our results revealed that the kinetics emerging from neuromodulatory activation of the signaling network were required to account for the dynamical changes in excitability. In summary, our integrated multiscale model provides, to our knowledge, a new approach for quantitative investigation of neuromodulatory effects on signaling and electrophysiology.