Voltage-dependent Ionic Currents Research Articles

Paracrine control of \u03b1-cell glucagon exocytosis is compromised in human type-2 diabetes

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.

SNC80 and naltrindole modulate voltage-dependent sodium, potassium and calcium channels via a putatively delta opioid receptor-independent mechanism.

SNC80 was designed as a highly selective nonpeptide delta opioid receptor (DOR) agonist. Antidepressant-like and antinociceptive effects of this compound were demonstrated in animal models. Naltrindole was synthetized as a highly selective DOR antagonist. Its antitussive and antinociceptive effects were reported. Observed effects of SNC80 and naltrindole may be accompanied by changes in neuronal excitability including modulation of voltage-dependent ion channels. We investigated possible DOR-independent modulation of neuronal sodium, calcium and potassium currents by both agents. NG108-15 cells lacking expression of DOR protein were used as model of neuronal cells. Cells were differentiated into neuronal phenotype by exposure to dibutyryl cyclic-AMP (dbcAMP). Lack of DORs expression in NG108-15 cells and the presence of DOR expression in brain and neuronal cultures were demonstrated by Western blot analysis. Both SNC80 and naltrindole exerted low to moderate modulatory effects on voltage-dependent ion currents. SNC80 weakly inhibited sodium current, potentiated calcium current, and did not act on potassium channels. Naltrindole inhibited sodium current, did not act on calcium current and inhibited potassium current at a high concentration. Such effects should be taken into account when these compounds are used for investigation of DOR-mediated signaling pathways.

Altered Sodium and Potassium, but not Calcium Currents in Cerebellar Granule Cells in an In Vitro Model of Neuronal Injury.

Acute injury of central nervous system (CNS) starts a cascade of morphological, molecular, and functional changes including formation of a fibrotic scar, expression of transforming growth factor beta 1 (TGF-β1), and expression of extracellular matrix proteins leading to arrested neurite outgrowth and failed regeneration. We assessed alteration of electrophysiological properties of cerebellar granule cells (CGCs) in two in vitro models of neuronal injury: (i) model of fibrotic scar created from coculture of meningeal fibroblasts and cerebral astrocytes with addition of TGF-β1; (ii) a simplified model based on administration of TGF-β1 to CGCs culture. Both models reproduced suppression of neurite outgrowth caused by neuronal injury, which was equally restored by chondroitinase ABC (ChABC), a key disruptor of fibrotic scar formation. Voltage-dependent calcium current was not affected in either injury model. However, intracellular calcium concentration could be altered as an expression of inositol trisphosphate receptor type 1 was suppressed by TGF-β1 and restored by ChABC. Voltage-dependent sodium current was significantly suppressed in CGCs cultured on a model of fibrotic scar and was only partly restored by ChABC. Administration of TGF-β1 significantly shifted current-voltage relation of sodium current toward more positive membrane potential without change to maximal current amplitude. Both transient and sustained potassium currents were significantly suppressed on a fibrotic scar and restored by ChABC to their control amplitudes. In contrast, TGF-β1 itself significantly upregulated transient and did not change sustained potassium current. Observed changes of voltage-dependent ion currents may contribute to known morphological and functional changes in injured CNS.

Voltage sensitive currents and information processing by single neurons

This is a computational study using data and models. We study selected neurons in neural circuits, which have different precision of spike timing under reproducible experimental conditions. Voltage sensitive currents govern foremost the spike generation at given times. Yet the effects of voltage sensitive currents are not limited to the axonal hillock, other neuronal structures are also involved. We compare previously described neural computations using models ranked according to level of detail and complexity. Single neurons propagate neural signal by following steps. The first is the synaptic processing, realized by function of pre- and post- synaptic membrane. Next is signal recoding and encoding and information processing, which is realized by dendrites. As next step follows binary decision spike generation with yes-or-no logical value. This occurs at the axonal hillock. Further signal recoding and encoding is computed by axonal propagation. Finally there is again synaptic processing at the synapse to the next neuron. In past, some salient features amongst these steps were implemented in biophysically detailed neuronal simulations of anatomically reconstructed neurons. This was achieved via brute-force numerical solution of many parallel instances of cable equation solved by numerical solver of partial differential equations named GENESIS [1]. The experimental observation of voltage dependent ion currents (active currents) in dendrites has been a milestone confirming high complexity and high information throughput in single neurons [2]. We use computational description of the pyramidal neuron CA3 and the MSO neuron. The CA3 is anatomical acronym of Cornu Ammonis, 3rd area of hippocampus and the MSO, medial superior olive is a nucleus in the binaural auditory pathway. These model neurons are embodied into three implementations differing by their levels of complexity, as follows. 1. The first level (macroscopic) unit is phenomenological model with black-box components containing elementary arithmetic units connected together, generating spikes as uniform, unitary events [3]. 2. The second level (mesoscopic) unit is medium complexity model with delays and voltages described in experiments [4]. 3. The third level (microscopic) unit is biophysically realistic detailed model based on the anatomical reconstruction of single neuron [1]. In the three levels we describe necessary time step duration and computational complexity, which has as its practical consequence ration of simulated versus real time on a prototypical contemporary personal computer [5,6]. The implementation details may vary. We use GCC, gcc.gnu.org; www.scholarpedia.org/article/GENESIS[1]; MATLAB (TM), www.mathworks.com; octave, wiki.octave.org[3]; and other software and libraries. In order to abstract the implementation details, we theoretically estimate and recommend the useful range of time step sizes, number of compartments and computational complexity. This enables us to obtain asymptotic and converging figures of information transfer rates and therefore a measure of computational complexity, obtained by the units of three different levels. Finally, we analyze several models in computational neuroscience literature (including our previous works). Based on this analysis, we discuss whether the computational resources were used efficiently.

Abstract 12087: KCa3.1 Channels Support Resting Potential and Proliferative Function During Store-Operated Ca 2+ -Entry in c-kit + Endogenous Cardiac Stem Cells

Introduction: Adult hearts contain c-kit + endogenous cardiac stem cells (eCSCs). Cultured eCSCs express a wide range of ion channels, but cell culture is known to affect phenotype. Here we defined ion currents in freshly isolated c-kit + eCSCs and their potential functional role. Methods: eCSCs isolated from healthy and heart failure (HF, ventricular tachypacing X 2 wks) dog hearts were magnetically purified with c-kit antibodies. Ion currents and membrane potential were recorded with patch clamp. Results: Contrary to cultured c-kit + eCSCs, ion currents were barely detectable with Ca 2+ i buffered (Fig A). Free Ca 2+ i activated prominent I KCa3.1 (TRAM34 sensitive, Fig B) in 82% of cells. I KCa3.1 was reduced in eCSCs isolated from HF dogs (Fig C), along with corresponding KCNN4 mRNA (Fig D). Under perforated patch to maintain spontaneous physiological [Ca 2+ ] i , eCSCs had a membrane potential (V mem ) of -73±10 mV. Ca 2+ i depletion with nominally 0 mM [Ca 2+ ] o strongly depolarized the membrane. After passive Ca 2+ i depletion, store-operated channel (SOC) mediated Ca 2+ entry (SOCE) caused a robust V mem hyperpolarization (from -21±4 to -79±7 mV, p<0.001). The SOCE effect on V mem was substantially reduced by both 2-APB (SOC inhibitor) and TRAM34 (Fig E). Inhibiting KCa3.1 (TRAM34, 0.1 μM) or SOCs (2-APB, 50 μM) in culture decreased c-kit + eCSCs proliferation by 21% (p<0.05) and 55% (p<0.001) respectively. Conclusions: In contrast to cultured eCSCs, voltage-dependent ion currents are virtually absent from freshly isolated c-kit + eCSCs. I KCa3.1 is prominent and maintains V mem in the face of substantial Ca 2+ entry from SOCs. This Ca 2+ entry would normally depolarize the cell, reduce the gradient for Ca 2+ entry and turn off the Ca 2+ signal. We hypothesize that the Ca 2+ -mediated KCa3.1 K + -conductance increase is necessary to maintain V mem in the face of the Ca 2+ entry that activates eCSC proliferation, and that KCa3.1 channels may therefore play an important role in eCSC function.

Selective Gating of Neuronal Activity by Intrinsic Properties in Distinct Motor Rhythms.

Many neural circuits show fast reconfiguration following altered sensory or modulatory inputs to generate stereotyped outputs. In the motor circuit of Xenopus tadpoles, I study how certain voltage-dependent ionic currents affect firing thresholds and contribute to circuit reconfiguration to generate two distinct motor patterns, swimming and struggling. Firing thresholds of excitatory interneurons [i.e., descending interneurons (dINs)] in the swimming central pattern generator are raised by depolarization due to the inactivation of Na(+) currents. In contrast, the thresholds of other types of neurons active in swimming or struggling are raised by hyperpolarization from the activation of fast transient K(+) currents. The firing thresholds are then compared with the excitatory synaptic drives, which are revealed by blocking action potentials intracellularly using QX314 during swimming and struggling. During swimming, transient K(+) currents lower neuronal excitability and gate out neurons with weak excitation, whereas their inactivation by strong excitation in other neurons increases excitability and enables fast synaptic potentials to drive reliable firing. During struggling, continuous sensory inputs lead to high levels of network excitation. This allows the inactivation of Na(+) currents and suppression of dIN activity while inactivating transient K(+) currents, recruiting neurons that are not active in swimming. Therefore, differential expression of these currents between neuron types can explain why synaptic strength does not predict firing reliability/intensity during swimming and struggling. These data show that intrinsic properties can override fast synaptic potentials, mediate circuit reconfiguration, and contribute to motor-pattern switching.

Optogenetic Manipulation of Ca2+ Transients and Contraction in a Mathematical Model of Cardiomyocyte Function

The cardiac optogenetics ‘toolbox’ combines optical physics and genetic engineering technology to provide a means of manipulating heart cell function with high specificity. For instance, recent studies have shown that light irradiance at specific wavelengths can perturb the processes of excitation, transmembrane potential propagation, and beating frequency in ChR2-expressing cardiac cells. We hypothesized that optogenetics could be used to manipulate not just beating frequency of heart cells, but also the intensity of contractions.In this study, we present an integrative optogenetics-electromechanical mathematical model that uses an engineered light-electric impulse to stimulate genetically modified cardiomyocytes. This hybrid excitation protocol simulates the excess inward ionic current produced by brief, high-intensity light flashes and demonstrates that when properly timed, these pulses can be used to manipulate the cellular action potential duration, intracellular calcium release, and myofilament twitch dynamics. The model is composed of three coupled modules; a light controlled ion channel (ChR2) module, an electrical cellular model with enhanced calcium induced calcium released process (CICR), and a myofilament (MF) mechanical component. The cell membrane connects activity of ChR2 with other time- and voltage-dependent ionic currents in the cell as well as CICR. The Ca2+ transient produced by CICR then links ChR2 activity to the local cardiac MF dynamics causing muscle to contract. The model is expected to be useful for designing many different types of in vitro cardiomyocytes studies where beat-to-beat manipulation of contractility is desirable.

Complex Early and Delayed Afterdepolarization Dynamics caused by Voltage-Calcium Coupling in Cardiac Myocytes

Please cite this article in press as: Song et al., Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes, Bio- physical Journal (2015), http://dx.doi.org/10.1016/j.bpj.2015.03.011 Biophysical Journal Volume 108 April 2015 1–14 Article Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes Zhen Song, 1,2 Christopher Y. Ko, 1,2 Michael Nivala, 1,2 James N. Weiss, 1,2,3 and Zhilin Qu 1,2, * Cardiovascular Research Laboratory, 2 Department of Medicine (Cardiology), and 3 Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California ABSTRACT Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other’s behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of I Ca,L ; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., calcium-wave-dependent and a calcium-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable. INTRODUCTION Early afterdepolarizations (EADs) and delayed afterdepola- rizations (DADs) are known to cause arrhythmias in a vari- ety of cardiac diseases, including long QT syndromes (1–3), catecholaminergic polymorphic ventricular tachycardia (CPVT) (4,5), and heart failure (6,7). EADs are voltage oscillations that occur during the repolarizing phases of the action potential (AP). By prolonging the AP duration (APD) regionally, EADs can increase dispersion of refracto- riness, forming a tissue substrate vulnerable to reentry. Furthermore, if EADs reach the threshold to propagate out this region as premature ventricular contractions, they can serve as triggers to induce reentry. DADs, on the other hand, are voltage oscillations during diastole. By depolariz- ing the resting membrane potential regionally, DADs can cause conduction block (8), and, if they reach the threshold for sodium (Na) channel activation, can generate triggered activity (TA) manifesting as premature ventricular contrac- tions that induce reentry. EADs occur in the setting of reduced repolarization reserve, such as long QT syndromes, and have classically been attributed to voltage-driven oscillations in the repola- rizing phase of the AP, although spontaneous sarcoplasmic Submitted October 29, 2014, and accepted for publication March 10, 2015. *Correspondence: zqu@mednet.ucla.edu reticulum (SR) calcium (Ca) release has also been proposed as an important mechanism. DADs, on the other hand, are promoted by Ca cycling disorders, such as CPVT and digi- talis toxicity, which promote Ca-driven oscillations. Heart failure represents a combination of reduced repolarization reserve and abnormal Ca cycling resulting from electrical and excitation-contraction coupling remodeling processes driven by both genetic transcriptional and posttranslational signaling components. In all of these settings, it is common for EADs and DADs to coexist and influence each other. This is because membrane voltage is strongly affected by Ca-sensitive ionic currents, and, conversely, cellular Ca loading is strongly influenced by voltage-dependent ionic currents, referred to as bidirectional Ca-voltage coupling. Ca-voltage coupling can promote complex AP dynamics in the heart (9). Analyzing the interactions between EADs and DADs (and voltage and Ca-cycling coupling dynamics in general), however, has been challenging, because the Ca cycling dynamics (e.g., Ca waves and oscillations) result from a spatially distributed heterogeneous network of Ca release units (CRUs) in the cell. The Ca waves emanating from various subcellular regions integrate to depolarize dia- stolic membrane voltage by activating Ca-sensitive inward currents including the Na-Ca exchange (NCX) current (I NCX ) and Ca-activated nonselective cation channels (I ns(Ca) ), generating DADs. Similarly, spontaneous SR Ca Editor: Randall Rasmusson. O 2015 by the Biophysical Society http://dx.doi.org/10.1016/j.bpj.2015.03.011 BPJ 6468

The role of linear and voltage-dependent ionic currents in the generation of slow wave oscillations

Neuronal oscillatory activity is generated by a combination of ionic currents, including at least one inward regenerative current that brings the cell towards depolarized voltages and one outward current that repolarizes the cell. Such currents have traditionally been assumed to require voltage-dependence. Here we test the hypothesis that the voltage dependence of the regenerative inward current is not necessary for generating oscillations. Instead, a current I NL that is linear in the biological voltage range and has negative conductance is sufficient to produce regenerative activity. The current I NL can be considered a linear approximation to the negative-conductance region of the current-voltage relationship of a regenerative inward current. Using a simple conductance-based model, we show that I NL , in conjunction with a voltage-gated, non-inactivating outward current, can generate oscillatory activity. We use phase-plane and bifurcation analyses to uncover a rich variety of behaviors as the conductance of I NL is varied, and show that oscillations emerge as a result of destabilization of the resting state of the model neuron. The model shows the need for well-defined relationships between the inward and outward current conductances, as well as their reversal potentials, in order to produce stable oscillatory activity. Our analysis predicts that a hyperpolarization-activated inward current can play a role in stabilizing oscillatory activity by preventing swings to very negative voltages, which is consistent with what is recorded in biological neurons in general. We confirm this prediction of the model experimentally in neurons from the crab stomatogastric ganglion.

Contribution of near-threshold currents to intrinsic oscillatory activity in rat medial entorhinal cortex layer II stellate cells

The temporal lobe is well known for its oscillatory activity associated with exploration, navigation, and learning. Intrinsic membrane potential oscillations (MPOs) and resonance of stellate cells (SCs) in layer II of the entorhinal cortex are thought to contribute to network oscillations and thereby to the encoding of spatial information. Generation of both MPOs and resonance relies on the expression of specific voltage-dependent ion currents such as the hyperpolarization-activated cation current (I(H)), the persistent sodium current (I(NaP)), and the noninactivating muscarine-modulated potassium current (I(M)). However, the differential contributions of these currents remain a matter of debate. We therefore examined how they modify neuronal excitability near threshold and generation of near-threshold MPOs and resonance in vitro. We found that resonance mainly relied on I(H) and was reduced by I(H) blockers and modulated by cAMP and an I(M) enhancer but that neither of the currents exhibited full control over MPOs in these cells. As previously reported, I(H) controlled a theta-frequency component of MPOs such that blockade of I(H) resulted in fewer regular oscillations that retained low-frequency components and high peak amplitude. However, pharmacological inhibition and augmentation of I(M) also affected MPO frequencies and amplitudes. In contrast to other cell types, inhibition of I(NaP) did not result in suppression of MPOs but only in a moderation of their properties. We reproduced the experimentally observed effects in a single-compartment stochastic model of SCs, providing further insight into the interactions between different ionic conductances.

Ion currents and gap junction coupling in isolated radial glial cells

Event Abstract Back to Event Ion currents and gap junction coupling in isolated radial glial cells Márta Jelitai1*, Károly Markó1, Nóra Hádinger1 and Emília Madarász1 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary According to the accepted view, cell-physiological properties of stem and progenitor cells contribute to the maintenance of a non-differentiated status even in a highly differentiated environment, but help to respond to specific differentiation inducing signals. The cellular “excitability” and responsiveness to environmental stimuli are strongly influenced by the local changes in the intracellular distribution of ions. Changes in the expression and in the functional properties of ion channels play deterministic role in neuronal commitment and neuronal differentiation. In the current work, the characteristics of selectively isolated radial glial cells from embryonic or adult nerve tissue- were studied using dye spreading and patch-clamp techniques. Radial glial cells are less gap junction coupled than the neural stem cells and the rate of gap junction coupling changes dynamically. According to the level of gap junction coupling radial glial cells display passive conductance. Delayed-rectifying outward potassium current was expressed almost in all isolated radial glial cell, however the pharmacological properties of the current changed significantly with development. In cells with neuronal morphology voltage dependent sodium current was recorded. The characterization of voltage-dependent ion currents and their developmental appearance may reveal some regulatory roles of ion channel functions in the maintenance of the progenitor stage. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Jelitai M, Markó K, Hádinger N and Madarász E (2010). Ion currents and gap junction coupling in isolated radial glial cells. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00071 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2010; Published Online: 21 Apr 2010. * Correspondence: Márta Jelitai, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, jelitai@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Márta Jelitai Károly Markó Nóra Hádinger Emília Madarász Google Márta Jelitai Károly Markó Nóra Hádinger Emília Madarász Google Scholar Márta Jelitai Károly Markó Nóra Hádinger Emília Madarász PubMed Márta Jelitai Károly Markó Nóra Hádinger Emília Madarász Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The Role of Ion Channels in Differentiating Chondrocytes

Cartilage is an important load-bearing component of the skeleton of vertebrates. This tissue has similar composition to other members of the connective tissue family: major constituents are cells, the chondrocytes, and the surrounding extracellular matrix; however, it is also unique being avascular. Chondrocytes are non-excitable cells and little is known about their plasma membrane ion channels. The aim of our study was to identify ion channels and establish their roles in differentiating chondrocytes.Our in vitro chondrogenesis model system is a high density mesenchymal cell culture, in which chondroprogenitor cells are isolated from limb buds of chicken embryos. Using whole-cell patch-clamp we have detected voltage-dependent ionic currents in these differentiating cells, whose amplitude depended on the time elapsed since isolation. An outward current was present in chondrocytes within 1-2 days of isolation, while an inward current gradually replaced it about 2 days after isolation. Using ion substitution experiments we identified the channels responsible for the currents as voltage-gated K+ and Na+ channels, respectively.The average amplitude of the Na+ current in cells during days 3-4 following isolation was −294 +/− 22 pA at 0 mV. The current inactivated with a time constant of tau = 0.59 +/− 0.04 ms. The voltage-dependence of steady state activation and inactivation were also determined yielding V1/2 values of −38 and −72 mV. Tetrodotoxin reversibly blocked the current with a Kd = 12 nM. The results of planned molecular biological experiments combined with our biophysical and pharmacological data will be used to identify the channel. The characterization of the K+ channel is presently underway.In many cell types changes in ion channel expression are associated with differentiation, thus our long-term aim is the clarification of the role of these channels in chondrogenesis and its potential clinical consequences.

Dopamine D1/5 Receptor-Mediated Long-Term Potentiation of Intrinsic Excitability in Rat Prefrontal Cortical Neurons: Ca2+-Dependent Intracellular Signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.

Distribution of Kv1‐like potassium channels in the electromotor and electrosensory systems of the weakly electric fish Apteronotus leptorhynchus

The electromotor and electrosensory systems of the weakly electric fish Apteronotus leptorhynchus are model systems for studying mechanisms of high-frequency motor pattern generation and sensory processing. Voltage-dependent ionic currents, including low-threshold potassium currents, influence excitability of neurons in these circuits and thereby regulate motor output and sensory filtering. Although Kv1-like potassium channels are likely to carry low-threshold potassium currents in electromotor and electrosensory neurons, the distribution of Kv1 alpha subunits in A. leptorhynchus is unknown. In this study, we used immunohistochemistry with six different antibodies raised against specific mammalian Kv1 alpha subunits (Kv1.1-Kv1.6) to characterize the distribution of Kv1-like channels in electromotor and electrosensory structures. Each Kv1 antibody labeled a distinct subset of neurons, fibers, and/or dendrites in electromotor and electrosensory nuclei. Kv1-like immunoreactivity in the electrosensory lateral line lobe (ELL) and pacemaker nucleus are particularly relevant in light of previous studies suggesting that potassium currents carried by Kv1 channels regulate neuronal excitability in these regions. Immunoreactivity of pyramidal cells in the ELL with several Kv1 antibodies is consistent with Kv1 channels carrying low-threshold outward currents that regulate spike waveform in these cells (Fernandez et al., J Neurosci 2005;25:363-371). Similarly, Kv1-like immunoreactivity in the pacemaker nucleus is consistent with a role of Kv1 channels in spontaneous high-frequency firing in pacemaker neurons. Robust Kv1-like immunoreactivity in several other structures, including the dorsal torus semicircularis, tuberous electroreceptors, and the electric organ, indicates that Kv1 channels are broadly expressed and are likely to contribute significantly to generating the electric organ discharge and processing electrosensory inputs.

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG.

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl- current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.

Voltage- and calcium-activated currents in cultured olfactory receptor neurons of male Mamestra brassicae (Lepidoptera).

Insect olfactory receptor neurons (ORNs) grown in primary cultures were studied using the patch-clamp technique in both conventional and amphotericin B perforated whole-cell configurations under voltage-clamp conditions. After 10-24 days in vitro, ORNs had a mean resting potential of -62 mV and an average input resistance of 3.2 GOmega. Five different voltage-dependent ionic currents were isolated: one Na(+), one Ca(2+) and three K(+) currents. The Na(+) current (35-300 pA) activated between -50 and -30 mV and was sensitive to 1 microM tetrodotoxin (TTX). The sustained Ca(2+) current activated between -30 and -20 mV, reached a maximum amplitude at 0 mV (-4.5 +/- 6.0 pA) that increased when Ba(2+) was added to the bath and was blocked by 1 mM Co(2+). Total outward currents were composed of three K(+) currents: a Ca(2+)-activated K(+) current activated between -40 and -30 mV and reached a maximum amplitude at +40 mV (605 +/- 351 pA); a delayed-rectifier K(+) current activated between -30 and -10 mV, had a mean amplitude of 111 +/- 67 pA at +60 mV and was inhibited by 20 mM tetraethylammonium (TEA); and, finally, more than half of ORNs exhibited an A-like current strongly dependent on the holding potential and inhibited by 5 mM 4-aminopyridine (4-AP). Pheromone stimulation evoked inward current as measured by single channel recordings.

Reliable, responsive pacemaking and pattern generation with minimal cell numbers: the crustacean cardiac ganglion.

Investigations of the electrophysiology of crustacean cardiac ganglia over the last half-century are reviewed for their contributions to elucidating the cellular mechanisms and interactions by which a small (as few as nine cells) neuronal network accomplishes extremely reliable, rhythmical, patterned activation of muscular activity-in this case, beating of the neurogenic heart. This ganglion is thus a model for pacemaking and central pattern generation. Favorable anatomy has permitted voltage- and space-clamp analyses of voltage-dependent ionic currents that endow each neuron with the intrinsic ability to respond with rhythmical, patterned impulse activity to nonpatterned stimulation. The crustacean soma and initial axon segment do not support impulse generation but integrate input from stretch-sensitive dendrites and electrotonic and chemically mediated synapses on axonal processes in neuropils. The soma and initial axon produce a depolarization-activated, calcium-mediated, sustained potential, the "driver potential," so-called because it drives a train of impulses at the "trigger zone" of the axon. Extreme reliability results from redundancy and the electrotonic coupling and synaptic interaction among all the neurons. Complex modulation by central nervous system inputs and by neurohormones to adjust heart pumping to physiological demands has long been demonstrated, but much remains to be learned about the cellular and molecular mechanisms of action. The continuing relevance of the crustacean cardiac ganglion as a relatively simple model for pacemaking and central pattern generation is confirmed by the rapidly widening documentation of intrinsic potentials such as plateau potentials in neurons of all major animal groups. The suite of ionic currents (a slowly inactivating calcium current and various potassium currents, with variations) observed for the crustacean cardiac ganglion have been implicated in or proven to underlie a majority of the intrinsic potentials of neurons involved in pattern generation.

Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells.

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.

Low external NaCl concentration and low osmolarity enhance voltage-gated Ca currents but depress K currents in freshly isolated rat hippocampal neurons

In previous experiments, reducing bath osmolarity ( π o) or external NaCl concentration ([NaCl] o) caused an increase in synaptic currents recorded in whole-cell configuration from pyramidal cells of CA1 region of hippocampal slices. Slow inward current surges, assumed to be calcium currents, were also enhanced. This contrasted with the strong, reversible, generalized depression of voltage-dependent ion currents in isolated neurons, caused by sudden, brief exposure to very low π o. I have now recorded voltage-dependent whole-cell Na-, K- and Ca-currents from freshly isolated hippocampal CA1 pyramidal neurons during more gradual lowering of π o or [NaCl] o. Changes in cell size were determined from image areas and changes in intracellular Ca 2+ activity were measured as the ratio of the fluorescences of fluo-3/fura-red by confocal microscopy. Iso-osmotic substitution of 40 or 60 mM NaCl by mannitol or sucrose for 5–7 min, or reducing π o by deleting NaCl from the bath (osmolarity decreased by 69 or 108 mosM/kg) depressed K currents. Na currents were also strongly depressed, but this is in part attributable to reduced driving potential and ionic conductance. The depression of I K varied widely and it was inversely correlated with the degree of hypotonic swelling, suggesting that reduced ion channel conductance also reduces permeability to water. Reducing [NaCl] o by 60 mM, or π o by 105 mosM/kg consistently and reversibly increased Ca currents. Intracellular Ca 2+ level also increased, but the changes of [Ca 2+] i and I Ca were not correlated. Facilitation of the Ca influx into presynaptic terminals could explain the increase of synaptic currents. Depression of outward currents could also contribute to the irritability of the central nervous system typical of clinical hyponatremia.

Characterization of ionic currents of cells of the subfornical organ that project to the supraoptic nuclei

The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current.