Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

Coordinated glucose-induced Ca2+ and pH responses in yeast Saccharomyces cerevisiae.

Ca2+ and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca2+-pH link, cytosolic Ca2+ was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca2+ transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca2+ transients and vacuolar alkalinization. In external Ca2+-free medium, glucose-induced pH responses were not affected but Ca2+ transients were abolished, indicating that the intracellular [Ca2+] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca2+ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca2+ influx that is associated with glucose-induced Ca2+ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca2+ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H+/Ca2+ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H+/Ca2+ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca2+.

Investigating the Antifungal Mechanism of Action of Polygodial by Phenotypic Screening in Saccharomyces cerevisiae.

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.

Decreased Vacuolar Ca2+ Storage and Disrupted Vesicle Trafficking Underlie Alpha-Synuclein-Induced Ca2+ Dysregulation in S. cerevisiae.

The yeast Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.

A Micropatterned Multielectrode Shell for 3D Spatiotemporal Recording from Live Cells.

Microelectrode arrays (MEAs) have proved to be useful tools for characterizing electrically active cells such as cardiomyocytes and neurons. While there exist a number of integrated electronic chips for recording from small populations or even single cells, they rely primarily on the interface between the cells and 2D flat electrodes. Here, an approach that utilizes residual stress-based self-folding to create individually addressable multielectrode interfaces that wrap around the cell in 3D and function as an electrical shell-like recording device is described. These devices are optically transparent, allowing for simultaneous fluorescence imaging. Cell viability is maintained during and after electrode wrapping around the cel and chemicals can diffuse into and out of the self-folding devices. It is further shown that 3D spatiotemporal recordings are possible and that the action potentials recorded from cultured neonatal rat ventricular cardiomyocytes display significantly higher signal-to-noise ratios in comparison with signals recorded with planar extracellular electrodes. It is anticipated that this device can provide the foundation for the development of new-generation MEAs where dynamic electrode-cell interfacing and recording substitutes the traditional method using static electrodes.

Inhibition of glutamate decarboxylase (GAD) by ethyl ketopentenoate (EKP) induces treatment-resistant epileptic seizures in zebrafish.

Epilepsy is a chronic brain disorder characterized by recurrent seizures due to abnormal, excessive and synchronous neuronal activities in the brain. It affects approximately 65 million people worldwide, one third of which are still estimated to suffer from refractory seizures. Glutamic acid decarboxylase (GAD) that converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Importantly, clinical evidence shows that lowered GAD activity is associated with several forms of epilepsy which are often treatment resistant. In the present study, we synthetized and explored the possibility of using ethyl ketopentenoate (EKP), a lipid-permeable GAD-inhibitor, to induce refractory seizures in zebrafish larvae. Our results demonstrate that EKP evoked robust convulsive locomotor activities, excessive epileptiform discharges and upregulated c-fos expression in zebrafish. Moreover, transgenic animals in which neuronal cells express apoaequorin, a Ca2+-sensitive bioluminescent photoprotein, displayed large luminescence signals indicating strong EKP-induced neuronal activation. Molecular docking data indicated that this proconvulsant activity resulted from the direct inhibition of both gad67 and gad65. Limited protective efficacy of tested anti-seizure drugs (ASDs) demonstrated a high level of treatment resistance of EKP-induced seizures. We conclude that the EKP zebrafish model can serve as a high-throughput platform for novel ASDs discovery.

Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes.

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10-7 to 2.10-5M) and verapamil (7 concentrations from 10-11 to 10-5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8M and prolonged the APD with EC50 of 2.5·10-8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10-7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.

NKCC1 downregulation induces hyperpolarizing shift of GABA responsiveness at near term fetal stages in rat cultured dorsal root ganglion neurons.

BACKGROUND: GABAA receptor-mediated neurotransmission is greatly influenced by cation-chloride cotransporter activity during developmental stages. In embryonic neurons Na-K-2Cl (NKCC1) cotransporters mediate active chloride uptake, thus increasing the intracellular chloride concentration associated with GABA-induced depolarization. At fetal stages near term, oxytocin-induced NKCC1 downregulation has been implicated in the developmental shift from depolarizing to hyperpolarizing GABA action. Mature dorsal root ganglion neurons (DRGN), however, express high NKCC1 levels and maintain high intracellular chloride levels with consequent GABA-induced depolarization. RESULTS: Gramicidin-perforated patch-clamp recordings were used to assess the developmental change in chloride homeostasis in rat cultured small DRGN at the embryonic day 16 (E16) and 19 (E19). The results were compared to data previously obtained in fetal DRGN at E14 and in mature cells. A significant NKCC1 downregulation, leading to reduction in excitatory GABAergic transmission, was observed at E16 and E19. CONCLUSION: These results indicate that NKCC1 activity transiently decreases in DRGN at fetal stages near term. This developmental shift in GABAergic transmission may contribute to fetal analgesia and neuroprotection at birth.

Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage.

Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopathy.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3ß with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer's disease.

Primary culture of embryonic rat olfactory receptor neurons.

Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate.

Open-cell recording of action potentials using active electrode arrays.

The investigation of complex communication in cellular networks requires superior measurement tools than those available to date. Electrode arrays integrated onto silicon electronics are increasingly used to measure the electrical activity of cells in an automated and highly parallelized fashion, but they are restricted to recording extracellular potentials. Here, we report on an array of TiN electrodes built using standard silicon electronics for intracellular action potential recording. Intracellular access, possible at each of the 16 384 electrodes on the chip, was accomplished by local membrane electroporation using electrical stimulation with subcellular, micrometer-sized electrodes. Access to the cell interior was transient and could be tuned in duration by adapting the electroporation protocol. Intracellular sensing was found to be minimally invasive in the short and long-term, allowing consecutive intracellular recordings from the same cell over the course of days. Finally, we applied this method to investigate the effect of an ion channel blocker on cardiac electrical activity. This technique opens the door to massively parallel, long-term intracellular recording for fundamental electrophysiology and drug screening.

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 µm CMOS chip.

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 µm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.

Mitochondrial dysfunction in familial amyotrophic lateral sclerosis.

A growing body of evidence suggests that mitochondrial dysfunctions play a crucial role in the pathogenesis of various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting both upper and lower motor neurons. Although ALS is predominantly a sporadic disease, approximately 10% of cases are familial. The most frequent familial form is caused by mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). A dominant toxic gain of function of mutant SOD1 has been considered as the cause of the disease and mitochondria are thought to be key players in the pathogenesis. However, the exact nature of the link between mutant SOD1 and mitochondrial dysfunctions remains to be established. Here, we briefly review the evidence for mitochondrial dysfunctions in familial ALS and discuss a possible link between mutant SOD1 and mitochondrial dysfunction.

Synaptic dysfunction in hippocampus of transgenic mouse models of Alzheimer's disease: a multi-electrode array study.

APP.V717I and Tau.P301L transgenic mice develop Alzheimer's disease pathology comprising important aspects of human disease including increased levels of amyloid peptides, cognitive and motor impairment, amyloid plaques and neurofibrillary tangles. The combined model, APP.V717I×Tau.P301L bigenic mice (biAT mice) exhibit aggravated amyloid and tau pathology with severe cognitive and behavioral defects. In the present study, we investigated early changes in synaptic function in the CA1 and CA3 regions of acute hippocampal slices of young APP.V717I, Tau.P301L and biAT transgenic animals. We have used planar multi-electrode arrays (MEA) and improved methods for simultaneous multi-site recordings from two hippocampal sub-regions. In the CA1 region, long-term potentiation (LTP) was severely impaired in all transgenic animals when compared with age-matched wild-type controls, while basal synaptic transmission and paired-pulse facilitation were minimally affected. In the CA3 region, LTP was normal in Tau.P301L and APP.V717I but clearly impaired in biAT mice. Surprisingly, frequency facilitation in CA3 was significantly enhanced in Tau.P301L mice, while not affected in APP.V717I mice and depressed in biAT mice. The findings demonstrate important synaptic changes that differ considerably in the hippocampal sub-regions already at young age, well before the typical amyloid or tau pathology is evident.

Depletion of PINK1 affects mitochondrial metabolism, calcium homeostasis and energy maintenance.

Loss-of-function mutations in the gene encoding the mitochondrial PTEN-induced putative kinase 1 (PINK1) are a major cause of early-onset familial Parkinson's disease (PD). Recent studies have highlighted an important function for PINK1 in clearing depolarized mitochondria by mitophagy. However, the role of PINK1 in mitochondrial and cellular functioning in physiological conditions is still incompletely understood. Here, we investigate mitochondrial and cellular calcium (Ca(2+)) homeostasis in PINK1-knockdown and PINK1-knockout mouse cells, both in basal metabolic conditions and after physiological stimulation, using unbiased automated live single-cell imaging in combination with organelle-specific fluorescent probes. Our data reveal that depletion of PINK1 induces moderate fragmentation of the mitochondrial network, mitochondrial membrane depolarization and increased production of reactive oxygen species. This results in reduced uptake of Ca(2+) by mitochondria after physiological stimulation. As a consequence, cells with knockdown or knockout of PINK1 display impaired mitochondrial ATP synthesis, which is exacerbated under conditions of increased ATP demand, thereby affecting cytosolic Ca(2+) extrusion. The impairment in energy maintenance was confirmed in the brain of PINK1-knockout mice by in vivo bioluminescence imaging. Our findings demonstrate a key role for PINK1 in the regulation of mitochondrial homeostasis and energy metabolism under physiological conditions.

G37R SOD1 mutant alters mitochondrial complex I activity, Ca(2+) uptake and ATP production.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the molecular mechanisms whereby these mutations induce motor neuron death remain controversial. Here, we show that stable overexpression of mutant human SOD1 (G37R) - but not wild-type SOD1 (wt-SOD1) - in mouse neuroblastoma cells (N2a) results in morphological abnormalities of mitochondria accompanied by several dysfunctions. Activity of the oxidative phosphorylation complex I was significantly reduced in G37R cells and correlated with lower mitochondrial membrane potential and reduced levels of cytosolic ATP. Using targeted chimeric aequorin we further analyzed the consequences of mitochondrial dysfunction on cellular Ca(2+) handling. Mitochondrial Ca(2+) uptake, elicited by IP(3)-induced Ca(2+) release from endoplasmic reticulum (ER) was significantly reduced in G37R cells, while uptake induced by a brief Ca(2+) pulse was not affected in permeabilized cells. The decreased mitochondrial Ca(2+) uptake resulted in increased cytosolic Ca(2+) transients, whereas ER Ca(2+) load and resting cytosolic Ca(2+) levels were not affected. Together, these findings suggest that the mechanism linking mutant G37R SOD1 and ALS involves mitochondrial respiratory chain deficiency resulting in ATP loss and impairment of mitochondrial and cytosolic Ca(2+) homeostasis.

Single-cell stimulation and electroporation using a novel 0.18 µ CMOS chip with subcellular-sized electrodes.

In drug screening and pharmaceutical research, high-throughput systems that are able to perform single-cell measurements are highly desired. Micro-electrode arrays try to answer this need but still suffer from significant drawbacks such as a small amount of electrodes and the inability to address single cells. Here, we present a novel multi-transistor array chip with 16,384 subcellular-sized electrodes based on 0.18 µm CMOS technology. We show that single-cell stimulation is possible by applying voltage pulses on the electrode to stimulate the cells lying on top. Electroporation of the cell membrane is observed using the whole-cell patch clamp technique and fluorescent dye-based live imaging. This technology could be used for high-throughput, single-cell manipulations for the purpose of large-scale drug screening and the investigation of fundamental cell processes.

Neurotoxicity of Alzheimer's disease Aß peptides is induced by small changes in the Aß42 to Aß40 ratio.

The amyloid peptides Aß(40) and Aß(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aß(42) seems more pathogenic than Aß(40), the reason for this is not well understood. We show here that small alterations in the Aß(42):Aß(40) ratio dramatically affect the biophysical and biological properties of the Aß mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aß(42):Aß(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aß species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aß peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

Localized electrical stimulation of in vitro neurons using an array of sub-cellular sized electrodes.

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 µm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells.