Optoelectronic control of single cells using organic photocapacitors.

Optical control of the electrophysiology of single cells can be a powerful tool for biomedical research and technology. Here, we report organic electrolytic photocapacitors (OEPCs), devices that function as extracellular capacitive electrodes for stimulating cells. OEPCs consist of transparent conductor layers covered with a donor-acceptor bilayer of organic photoconductors. This device produces an open-circuit voltage in a physiological solution of 330 mV upon illumination using light in a tissue transparency window of 630 to 660 nm. We have performed electrophysiological recordings on Xenopus laevis oocytes, finding rapid (time constants, 50 µs to 5 ms) photoinduced transient changes in the range of 20 to 110 mV. We measure photoinduced opening of potassium channels, conclusively proving that the OEPC effectively depolarizes the cell membrane. Our results demonstrate that the OEPC can be a versatile nongenetic technique for optical manipulation of electrophysiology and currently represents one of the simplest and most stable and efficient optical stimulation solutions.

Biaryl sulfonamide motifs up- or down-regulate ion channel activity by activating voltage sensors.

Voltage-gated ion channels are key molecules for the generation of cellular electrical excitability. Many pharmaceutical drugs target these channels by blocking their ion-conducting pore, but in many cases, channel-opening compounds would be more beneficial. Here, to search for new channel-opening compounds, we screen 18,000 compounds with high-throughput patch-clamp technology and find several potassium-channel openers that share a distinct biaryl-sulfonamide motif. Our data suggest that the negatively charged variants of these compounds bind to the top of the voltage-sensor domain, between transmembrane segments 3 and 4, to open the channel. Although we show here that biaryl-sulfonamide compounds open a potassium channel, they have also been reported to block sodium and calcium channels. However, because they inactivate voltage-gated sodium channels by promoting activation of one voltage sensor, we suggest that, despite different effects on the channel gates, the biaryl-sulfonamide motif is a general ion-channel activator motif. Because these compounds block action potential-generating sodium and calcium channels and open an action potential-dampening potassium channel, they should have a high propensity to reduce excitability. This opens up the possibility to build new excitability-reducing pharmaceutical drugs from the biaryl-sulfonamide scaffold.

Rational Design of a Conductive Collagen Heart Patch.

Cardiovascular diseases, including myocardial infarction, are the cause of significant morbidity and mortality globally. Tissue engineering is a key emerging treatment method for supporting and repairing the cardiac scar tissue caused by myocardial infarction. Creating cell supportive scaffolds that can be directly implanted on a myocardial infarct is an attractive solution. Hydrogels made of collagen are highly biocompatible materials that can be molded into a range of shapes suitable for cardiac patch applications. The addition of mechanically reinforcing materials, carbon nanotubes, at subtoxic levels allows for the collagen hydrogels to be strengthened, up to a toughness of 30 J m-1 and a two to threefold improvement in Youngs' modulus, thus improving their viability as cardiac patch materials. The addition of carbon nanotubes is shown to be both nontoxic to stem cells, and when using single-walled carbon nanotubes, supportive of live, beating cardiac cells, providing a pathway for the further development of a cardiac patch.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Valproic acid phase shifts the rhythmic expression of Period2::Luciferase.

Valproic acid (VPA) is an anticonvulsant used to treat bipolar disorder, a psychiatric disease associated with disturbances in circadian rhythmicity. Little is known about how VPA affects circadian rhythms. The authors cultured tissues containing the master brain pacemaker for circadian rhythmicity, the suprachiasmatic nuclei (SCN), and skin fibroblasts from transgenic PERIOD2::LUCIFERASE (PER2::LUC) mice and studied the effect of VPA on the circadian PER2::LUC rhythm by measuring bioluminescence. VPA (1 mM) significantly phase advanced the PER2::LUC rhythm when applied at a time point corresponding to the lowest (trough, ~ZT 0) PER2::LUC expression but phase delayed the PER2::LUC rhythm when the drug was administered at the time of highest (peak, ~ZT 12) protein expression. In addition, it significantly increased the overall amplitude of PER2::LUC oscillations at time points at or close to ZT 12 but had no effect on period. Real-time PCR analyses on mouse and human fibroblasts revealed that expressions of other clock genes were increased after 2 h treatment with VPA. Because VPA is known to inhibit histone deacetylation, the authors treated cultures with an established histone deacetylation inhibitor, trichostatin A (TSA; 20 ng/mL), to compare the effect of VPA and TSA on molecular rhythmicity. They found that TSA had similar effects on the PER2::LUC rhythm as VPA. Furthermore, VPA and TSA significantly increased acetylation on histone H3 but in comparison little on histone H4. Lithium is another commonly used treatment for bipolar disorder. Therefore, the authors also studied the impact of lithium chloride (LiCl; 10 mM) on the PER2::LUC rhythm. LiCl delayed the phase, but in contrast to VPA and TSA, LiCl lengthened the PER2::LUC period and had no effect on histone acetylation. These results demonstrate that VPA can delay or advance the phase, as well as increase the amplitude, of the PERIOD2::LUCIFERASE rhythm depending on the circadian time of application. Furthermore, the authors show that LiCl delays the phase and lengthens the period of the PER2::LUC rhythm, confirming previous reports on circadian lithium effects. These different molecular effects may underlie differential chronotherapeutic effects of VPA and lithium.

Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells.

One of the earliest morphological changes occurring in apoptosis is cell shrinkage associated with an increased efflux of K(+) and Cl(-) ions. Block of K(+) or Cl(-) channels prevents cell shrinkage and death. Recently, we found evidences for the activation of a voltage-dependent anion channel in the plasma membrane (pl-VDAC) of a hippocampal cell line undergoing apoptosis. Nothing is known on pl-VDAC in apoptotic cell death of neural cells at different stages of differentiation. We have addressed this issue in primary cultures of differentiated hippocampal neurons and embryonic neural stem cells (NSCs). In control hippocampal neurons, pl-VDAC is closed but acts as an NADH-ferricyanide reductase, while in apoptotic neurons, pl-VDAC is opened and the enzymatic activity is increased. Anti-VDAC antibodies block pl-VDAC and prevent apoptosis, as well as the increase in enzymatic activity. Conversely, in NSCs, pl-VDAC is scarcely seen and there is no NADH-ferricyanide reductase activity. In agreement, anti-VDAC antibodies do not affect the apoptotic process. Instead, we find activation of a Na(+) channel that has low voltage dependency, a conductance of 26 pS, and is blocked by amiloride, which also prevents apoptosis. Thus, it appears that activation of pl-VDAC during apoptosis is a critical event in differentiated neurons, but not in NSCs.

Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons.

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.

Inhibition of hippocampal synaptic transmission by impairment of Ral function.

Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating that the synaptic release probability was reduced. Overexpression of a dominant negative Ral mutant, Ral A28N, caused a strong inhibition of autaptic responses, which was associated with a shift to facilitation in a majority (80%) of the neurons. These results indicate that Ral, along with at least one other non-Rab GTPase, participates in presynaptic regulation in hippocampal neurons.

Effects on synaptic activity in cultured hippocampal neurons by influenza A viral proteins.

Certain viruses can infect neurons and cause persistent infections with restricted expression of viral proteins. To study the consequences of such viral proteins on synaptic functions, the effects of two influenza A virus proteins, the nonstructural protein 1 (NS1) and the nucleoprotein (NP), were analyzed in cultures of rat hippocampal neurons. Transduction of the NS1 and NP proteins into the neurons was performed by applying the 11-amino acid peptide transduction domain (PTD) of human immunodeficiency virus (HIV) TAT coupled to the viral proteins. Neurons exposed to the NS1 and NP fusion proteins (NS1-PTD and NP-PTD, respectively) for 4 h were immunopositive for these proteins as diffuse cytoplasmic and nuclear distribution. After exposure for 48 h to NP-PTD, a punctate pattern of the immunolabel appeared in dendritic spinelike processes. Electrophysiologically, a reduction in both the frequency of spontaneous excitatory synaptic activity and in the amplitude of the miniature excitatory postsynaptic currents were recorded after exposing the hippocampal neurons to NP-PTD between 17 and 22 days in culture. These changes may reflect disturbances in postsynaptic functions. No such alterations in synaptic activities were recorded after exposure to NS1-PTD or to green fluorescent protein-PTD, which was used as a control. Based on these findings the authors hypothesize that the viral NP, by its localization to dendritic spinelike structures, interferes with the expression or anchoring of postsynaptic glutamate receptors and thereby disturbs synaptic functions. Thus a persistent viral infection in the brain may be associated with functional disturbances at the synaptic level.

Exposure to interferon-gamma during synaptogenesis increases inhibitory activity after a latent period in cultured rat hippocampal neurons.

Certain disorders of the nervous system may have their origin in disturbances in the development of synaptic connections and network structure that may not become overt until later in life. As inflammatory cytokines can influence synaptic activity in neuronal cultures, we analysed whether cytokine exposure during synaptogenesis can lead to imbalances in a neuronal network. Short-term application of interferon-gamma (IFN-gamma), but not tumour necrosis factor-alpha, during peak synaptogenesis (but not before or after) in Sprague-Dawley rat hippocampal cultures, caused both a decrease in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) and an increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs). These effects were only detected in recordings made weeks later. This was not due to a depression of glutamatergic synapses or to a change in the relative number of neurons containing glutamic acid decarboxylase (GAD). There was an increase in the average amplitude of miniature IPSCs, and in GAD-expressing neurons the amplitude of miniature EPSCs were larger as well as the responses to glutamate. This indicates that IFN-gamma-treatment induced increased inhibition via postsynaptic changes. These effects of IFN-gamma treatment were not observed when neuronal nitric oxide synthase was inhibited. Our study therefore shows that exposure to IFN-gamma during a restricted period of development, which coincides with the peak of excitatory synaptogenesis, can cause progressive changes in synaptic activity in the network. Thus, cytokine exposure at a critical period of development may constitute a 'hit-and-run' mechanism for certain nervous system disorders that become manifest after a latency period.

Persistence of viral RNA in the brain of offspring to mice infected with influenza A/WSN/33 virus during pregnancy.

Epidemiological studies have indicated an association between influenza A virus infections during fetal life and neuropsychiatric diseases. To study the potential for influenza A virus infections to cause nervous system dysfunctions, we describe a mouse model using intranasal instillation of the mouse neuroadapted influenza A/WSN/33 strain in pregnant mice. Viral RNA and nucleoprotein were detected in fetal brains and the viral RNA persisted for at least 90 days of postnatal life. We have, thus, obtained evidence for transplacental passage of influenza virus in mice and the persistence of viral components in the brains of these animals into young adulthood.