Resting Membrane Potential Of Neurons Research Articles

The Physiological Mechanisms of Transcranial Direct Current Stimulation to Enhance Motor Performance: A Narrative Review.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique that applies a stable, low-intensity (1-2 mA) direct current to modulate neuronal activity in the cerebral cortex. This technique is effective, simple to operate, affordable, and widely employed across various fields. tDCS has been extensively used in clinical and translational research, with growing applications in military and competitive sports domains. In recent years, the use of tDCS in sports science has garnered significant attention from researchers. Numerous studies have demonstrated that tDCS can enhance muscle strength, explosive power, and aerobic metabolism, reduce fatigue, and improve cognition, thereby serving as a valuable tool for enhancing athletic performance. Additionally, recent research has shed light on the physiological mechanisms underlying tDCS, including its modulation of neuronal resting membrane potential to alter cortical excitability, enhancement of synaptic plasticity to regulate long-term potentiation, modulation of neurovascular coupling to improve regional cerebral blood flow, and improvement of cerebral network functional connectivity, which activates and reinforces specific brain regions. tDCS also enhances the release of excitatory neurotransmitters, further regulating brain function. This article, after outlining the role of tDCS in improving physical performance, delves into its mechanisms of action to provide a deeper understanding of how tDCS enhances athletic performance and offers novel approaches and perspectives for physical performance enhancement.

Calcium-sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.

Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.638C > T; p.Arg213Gln (NUIGi063-A, NUIGi063-B, NUIGi063-C) and 3 healthy controls (NUIGi064-A, NUIGi064-B, NUIGi064-C)

KCNQ2 encodes the potassium-gated voltage channel Kv7.2, responsible for the M−current, which contributes to neuronal resting membrane potential. Pathogenic variants in KCNQ2 cause early onset epilepsies, developmental and epileptic encephalopathies. In this study, we generated three iPSC lines from dermal fibroblasts of a 5 year-old female patient with the KCNQ2 c.638C > T (p.Arg213Gln) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines were validated by confirming the targeted mutation, SNP karyotyping, STR analysis, pluripotent gene expression, differentiation capacity into three germ layers, and were free of transgene integration and Mycoplasma.

The sodium leak complex and friends: Identifying protein partners of the NALCN channelosome.

The resting membrane potential (RMP) of neurons and other excitable cells is a fundamental property which underpins all electrical signalling in the body. The predominant depolarising influence on the RMP in neurons is mediated by the Na+ leak channel (NALCN), which requires at least three accessory proteins (UNC79, UNC80, and FAM155A) to form a functional channelosome. Knocking out either NALCN or UNC79 in mice leads to neonatal death, and genetic variation NALCN or UNC80 in human patients leads to severe disease phenotypes including pronounced neurological and developmental defects.

Clinical Practice Guidelines for the Use of Transcranial Direct Current Stimulation in Psychiatry.

Clinical Practice Guidelines for the Use of Transcranial Direct Current Stimulation in Psychiatry.

Changes in the excitability of primary hippocampal neurons following exposure to 3.0 GHz radiofrequency electromagnetic fields

Exposures to radiofrequency electromagnetic fields (RF-EMFs, 100 kHz to 6 GHz) have been associated with both positive and negative effects on cognitive behavior. To elucidate the mechanism of RF-EMF interaction, a few studies have examined its impact on neuronal activity and synaptic plasticity. However, there is still a need for additional basic research that further our understanding of the underlying mechanisms of RF-EMFs on the neuronal system. The present study investigated changes in neuronal activity and synaptic transmission following a 60-min exposure to 3.0 GHz RF-EMF at a low dose (specific absorption rate (SAR) < 1 W/kg). We showed that RF-EMF exposure decreased the amplitude of action potential (AP), depolarized neuronal resting membrane potential (MP), and increased neuronal excitability and synaptic transmission in cultured primary hippocampal neurons (PHNs). The results show that RF-EMF exposure can alter neuronal activity and highlight that more investigations should be performed to fully explore the RF-EMF effects and mechanisms.

Contribution of KCNQ and TREK Channels to the Resting Membrane Potential in Sympathetic Neurons at Physiological Temperature.

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

Combining endocannabinoids with retigabine for enhanced M-channel effect and improved KV7 subtype selectivity.

Retigabine is unique among anticonvulsant drugs by targeting the neuronal M-channel, which is composed of KV7.2/KV7.3 and contributes to the negative neuronal resting membrane potential. Unfortunately, retigabine causes adverse effects, which limits its clinical use. Adverse effects may be reduced by developing M-channel activators with improved KV7 subtype selectivity. The aim of this study was to evaluate the prospect of endocannabinoids as M-channel activators, either in isolation or combined with retigabine. Human KV7 channels were expressed in Xenopus laevis oocytes. The effect of extracellular application of compounds with different properties was studied using two-electrode voltage clamp electrophysiology. Site-directed mutagenesis was used to construct channels with mutated residues to aid in the mechanistic understanding of these effects. We find that arachidonoyl-L-serine (ARA-S), a weak endocannabinoid, potently activates the human M-channel expressed in Xenopus oocytes. Importantly, we show that ARA-S activates the M-channel via a different mechanism and displays a different KV7 subtype selectivity compared with retigabine. We demonstrate that coapplication of ARA-S and retigabine at low concentrations retains the effect on the M-channel while limiting effects on other KV7 subtypes. Our findings suggest that improved KV7 subtype selectivity of M-channel activators can be achieved through strategically combining compounds with different subtype selectivity.

TRESK is a modality-specific brake on nociceptor excitability.

Background K+ channels are constitutively open at rest and play a critical role in neural function. The activity of these channels sets the resting membrane potential near the K+ equilibrium, around −90 mV, and thus decreases excitability. The two-pore domain potassium channels (K2P) comprise a diverse family of K+ channels that contribute to neuronal resting membrane potential (Enyedi & Czirjak, 2010; Gada & Plant, 2019). With its first member identified over 20 years ago (Lesage et al. 1996), this large family comprises 15 members with a high level of expression in sensory neurons (Gada & Plant, 2019). Indeed, sensory neurons of the dorsal root and trigeminal ganglia express different members of the K2P family, of which, TRESK, TREK-1, TREK-2 and TRAAK are highly expressed and present in pain-sensing neurons (i.e. nociceptors). Over the past few years, the role of these channels in pain sensitivity has started emerging, although more studies are needed to fully understand how they adjust pain thresholds. This is especially true for the TRESK channel: in preclinical models of neuropathic pain, nerve injury is associated with a decrease in TRESK expression, whereas that of other K2P channels remains unchanged. This decrease expression is assummed to reduce the ‘brake’ that nociceptors face during depolarization, thus facilitating the generation of action potentials in these neurons and pain hypersensitivity (Tulleuda et al. 2011). In this issue of The Journal of Physiology, Castellanos et al. (2020) report the use of a global TRESK knockout (KO) mouse to explore the role of this channel in somatosensation. Their recordings form nociceptors isolated from TRESK KO mice confirmed previous observations that, despite displaying weaker K+ current density, these neurons have a resting membrane potential that is very close to their wild-type (WT) littermates. This observation suggests that the TRESK channel has minimal contribution to the resting membrane potential. However, the neurons of KO mice show a significantly higher membrane resistance, which is probably responsible for their lower rheobase. They also have wider action potentials (AP) and smaller hyperpolarizing after-potentials, which indicates that TRESK contributes to the repolarizing phase of the AP. When the injected depolarizing currents exceeded the AP threshold, nociceptors of TRESK KO mice fired significantly more APs, indicating that the removal of TRESK increases the excitability of nociceptors. Castellanos et al. (2020) then used a Ca imaging approach to examine the sensitivity of nociceptors to common agonists. Although the responses to capsaicin and menthol did not change, responsiveness to the TRPA1 agonist allyl isothiocyanate (AITC) was significantly reduced. This observation was also present in behavioural experiments, where KO mice displayed significantly less nociceptive behaviours after intraplantar AITC injection. In behavioural experiments, Castellanos et al. (2020) demonstrate that TRESK functions to prevent C-fibres from being activated by low-intensity mechanical stimuli and its deletion results in mechanical hypersensitivity. Indeed, TRESK KO mice had significantly lower mechanical withdrawal thresholds. Castellanos et al. (2020) then examined the temperature sensitivity of nociceptors. As opposed to heat sensitivity, where the investigators failed to see any difference between KO and WT mice, their sensitivity to cold was significantly affected. In skin–nerve preparations, the distribution of C-fibre activation thresholds by cold stimuli indicated a shift toward warmer temperatures, indicating that, during cooling, these fibres would become activated sooner than C-fibres from their WT littermates. These observations were also transposed to behavioural studies, where the sensitivity of KO mice to a 2°C (noxious cold) cold plate test is significantly higher than that of WT mice. Finally, the role of TRESK was examined in preclinical models of persistent inflammatory pain, nerve cuff-induced neuropathic pain, or chemotherapy-induced neuropathic pain. All three models showed similar levels of sensitization between TRESK KO and WT mice. Overall, the study by Castellanos et al. (2020) adds to the body of knowledge on the role of TRESK channels in pain sensitivity, indicating that regulation of channel expression is an important factor in the control of sensory neuron excitability. Interestingly, removal of the channel produces modality-specific increases in sensitivity, with a hyper-responsiveness to mechanical and cold stimuli, yet no changes to heat stimuli. This could be explained in part by the predominant expression of TRESK in non-peptidergic nociceptors (Weir et al. 2019), which have been proposed to underlie mechanical pain (Cavanaugh et al. 2009). Indeed, in the trigeminal ganglia, the channel is present in 73% of isolectin B4-positive nociceptors and 29% of calcitonin gene-related peptide-positive nociceptors (Weir et al. 2019). The study by Castellanos et al. (2020) opens the door to the possibility of developing TRESK activators that would help control the excitability of nociceptors during chronic pain conditions. No competing interests declared. Sole author. No funding was received.

CIRBP Ameliorates Neuronal Amyloid Toxicity via Antioxidative and Antiapoptotic Pathways in Primary Cortical Neurons.

It is generally accepted that the amyloid β (Aβ) peptide toxicity contributes to neuronal loss and is involved in the initiation and progression of Alzheimer's disease (AD). Cold-inducible RNA-binding protein (CIRBP) is reported to be a general stress-response protein, which is induced by different stress conditions. Previous reports have shown the neuroprotective effects of CIRBP through the suppression of apoptosis via the Akt and ERK pathways. The objective of this study is to examine the effect of CIRBP against Aβ-induced toxicity in cultured rat primary cortical neurons and attempt to uncover its underlying mechanism. Here, MTT, LDH release, and TUNEL assays showed that CIRBP overexpression protected against both intracellular amyloid β- (iAβ-) induced and Aβ25-35-induced cytotoxicity in rat primary cortical neurons. Electrophysiological changes responsible for iAβ-induced neuronal toxicity, including an increase in neuronal resting membrane potentials and a decrease in K+ currents, were reversed by CIRBP overexpression. Western blot results further showed that Aβ25-35 treatment significantly increased the level of proapoptotic protein Bax, cleaved caspase-3, and cleaved caspase-9 and decreased the level of antiapoptotic factor Bcl-2, but were rescued by CIRBP overexpression. Furthermore, CIRBP overexpression prevented the elevation of ROS induced by Aβ25-35 treatment by decreasing the activities of oxidative biomarker and increasing the activities of key enzymes in antioxidant system. Taken together, our findings suggested that CIRBP exerted protective effects against neuronal amyloid toxicity via antioxidative and antiapoptotic pathways, which may provide a promising candidate for amyloid-based AD prevention or therapy.

Dopamine negatively modulates the NCA ion channels in C. elegans.

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Extracellular Potassium and Seizures: Excitation, Inhibition and the Role of Ih.

Seizure activity leads to increases in extracellular potassium concentration ([K[Formula: see text]]o), which can result in changes in neuronal passive and active membrane properties as well as in population activities. In this study, we examined how extracellular potassium modulates seizure activities using an acute 4-AP induced seizure model in the neocortex, both in vivo and in vitro. Moderately elevated [K[Formula: see text]]o up to 9[Formula: see text]mM prolonged seizure durations and shortened interictal intervals as well as depolarized the neuronal resting membrane potential (RMP). However, when [K[Formula: see text]]o reached higher than 9[Formula: see text]mM, seizure like events (SLEs) were blocked and neurons went into a depolarization-blocked state. Spreading depression was never observed as the blockade of ictal events could be reversed within 1-2[Formula: see text]min after the raised [K[Formula: see text]]o was changed back to control levels. This concentration-dependent dual effect of [K[Formula: see text]]o was observed using in vivo and in vitro mouse brain preparations as well as in human neocortical tissue resected during epilepsy surgery. Blocking the Ih current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, modulated the elevated [K[Formula: see text]]o influence on SLEs by promoting the high [K[Formula: see text]]o inhibitory actions. These results demonstrate biphasic actions of raised [K[Formula: see text]]o on neuronal excitability and seizure activity.

TDCS/tACS and plasticity

Non-invasive brain stimulation with transcranial direct or alternating currents (tDCS and tACS) modulates neuronal activity and excitability in the human brain. The primary effect of both techniques is assumed to be an alteration of neuronal resting membrane potential. tDCS induces tonic alterations, whereas tACS has oscillatory effects. After-effects include plasticity induction via tDCS, whose direction depend on the polarity of the target electrode, current flow direction in relation to neuronal orientation, and stimulation parameters. Originally, tACS was assumed to induce acute modulation of oscillatory brain activity without plasticity induction, but recent studies showed that stimulation at high frequencies also induces neuroplasticity. This lecture will give an overview about effects and mechanisms of both stimulation protocols.

T-types make your clock tick.

Voltage gated T-type calcium channels are important regulators of rhythmic activity in the mammalian nervous system. T-type channels are ideally suited towards regulating neuronal excitability for several reasons. First, their voltage dependent gating properties generate a ‘window current’ that allows them to become active near typical neuronal resting membrane potentials. Second, their hyperpolarization induced recovery from inactivation kinetics supports rebound burst activity in many types of neurons (Huguenard & Prince, 1994). Finally, these channels associate with, and regulate, the functions of both calcium activated and voltage gated potassium channels, which in turn shape neuronal firing properties (Turner & Zamponi, 2014). Besides regulating neuronal activity, T-type channels also contribute to low threshold exocytosis through physical coupling to the vesicle release machinery (Weiss et al. 2012). All of these aspects of T-type channel function are of direct relevance to an interesting new study by Yu and colleagues (Yu et al. 2015), reported in this issue of The Journal of Physiology, on the role of T-type channels in the pineal gland.

A framework for categorizing electrode montages in transcranial direct current stimulation.

Transcranial Direct Current Stimulation (tDCS) is a non-invasive brain stimulation technique that has been reintroduced in the last decade and is now mainly used as a cognitive modulator in human neuroscience research. tDCS delivers a weak direct current (usually up to 2 mA) over the scalp and creates a constant electric field in the brain which can lead to acute alterations of the excitability of cortical areas by its subthreshold depolarizing or hyperpolarizing effects on neuronal resting membrane potentials (Nitsche and Paulus, 2000). Beyond these acute effects, stimulation for some minutes results in neuroplastic after-effects, which can last for over 1 h after stimulation (Nitsche and Paulus, 2001). With repeated usage, longer lasting effects can be induced, which are in the range of late-phase plasticity (Monte-Silva et al., 2013). The neuroplastic effects resemble LTP- and LTD-like plasticity of glutamatergic synapses (Liebetanz et al., 2002; Nitsche et al., 2003a). Therefore, this technique allows us to study neuroplasticity of the human brain in a reversible manner and to modulate plasticity-related functions such as memory or learning, which critically depend on neuroplasticity, in healthy and clinical populations. Traditionally, one or more surface-positive (anode) and negative (cathode) electrodes are used to deliver current; one is positioned over the target area and the other one is put over another cranial (intracephalic) or extracranial (extracephalic) region of the body. These electrodes are usually called active and reference electrode respectively. However, these terms can be technically improper and should be replaced with other terms such as “target” and “return” electrodes, because the size and the place of a return electrode have an impact on its effects and thus it might not be physiologically inert. The return electrode can contribute directly—and not only via determination of electrical field orientation—to physiological effects when put over the cranium as well (Brunoni et al., 2012). Several studies have also shown antagonistic effects of stimulation on visual cortex (Antal et al., 2004; Accornero et al., 2007) and motor cortex (Nitsche and Paulus, 2000) dependent on return electrode position. In any case, the position of the return electrode will affect electrical field orientation, which is critical for the efficacy, and direction of the effects (Bikson et al., 2010; Kabakov et al., 2012). In both—extracephalic and intracephalic conditions—positive (cathode) and negative (anode) poles are conventionally physiologically distinguished according to their effects on excitability of the brain. Basically, cathodal stimulation has hyperpolarizing effects, which lead to inhibition of cortical activity, while anodal stimulation has excitatory effects (Nitsche et al., 2003b, 2008). It should be worth noting that although every neuron undergoes hyperpolarizing and depolarizing, the physiological effect depends more on axonal/soma polarization (Arlotti et al., 2012), hence the physical and physiological aspects can be dissociated. General effects on excitability, which were obtained primarily in the human motor cortex, might also switch, turning from excitatory to inhibitory or vice versa, dependent on stimulation parameters such as intensity, and duration (Batsikadze et al., 2013; Monte-Silva et al., 2013), and position of the return electrode (Antal et al., 2004; Accornero et al., 2007). With a rise in prevalence of studies using tDCS, protocols have become more complex and varieties of tDCS montages were introduced and are used in different labs. Despite this extending diversity of tDCS electrode montages, to our best knowledge, there is no consensus among researchers in this field on a systematic framework for categorizing electrode montages in a unified way. In this short article, we propose a framework for categorization of tDCS montages according to physical characteristics. This categorization is based on published studies until October 2014. Our main motivation to propose this framework is to unify the classifications of electrode montages in a simple way; there are nevertheless several other advantages of this categorization. First, different montages that are used to target a specific brain area such as dorsolateral prefrontal cortex (DLPFC) could have different effects; therefore providing a unified classification enables us to take these differences into account. Furthermore, this classification gives us a chance to explore other novel potentials for electrode montages that so far have remained untouched. Lastly, a unified systematic framework will be helpful for presenting study methods and for extracting data for systematic reviews and meta-analyses in a more practical way.

Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels.

BackgroundInorganic polyphosphate (polyP) is a highly charged polyanion capable of interacting with a number of molecular targets. This signaling molecule is released into the extracellular matrix by central astrocytes and by peripheral platelets during inflammation. While the release of polyP is associated with both induction of blood coagulation and astrocyte extracellular signaling, the role of secreted polyP in regulation of neuronal activity remains undefined. Here we test the hypothesis that polyP is an important participant in neuronal signaling. Specifically, we investigate the ability of neurons to release polyP and to induce neuronal firing, and clarify the underlying molecular mechanisms of this process by studying the action of polyP on voltage gated channels.ResultsUsing patch clamp techniques, and primary hippocampal and dorsal root ganglion cell cultures, we demonstrate that polyP directly influences neuronal activity, inducing action potential generation in both PNS and CNS neurons. Mechanistically, this is accomplished by shifting the voltage sensitivity of NaV channel activation toward the neuronal resting membrane potential, the block KV channels, and the activation of CaV channels. Next, using calcium imaging we found that polyP stimulates an increase in neuronal network activity and induces calcium influx in glial cells. Using in situ DAPI localization and live imaging, we demonstrate that polyP is naturally present in synaptic regions and is released from the neurons upon depolarization. Finally, using a biochemical assay we demonstrate that polyP is present in synaptosomes and can be released upon their membrane depolarization by the addition of potassium chloride.ConclusionsWe conclude that polyP release leads to increased excitability of the neuronal membrane through the modulation of voltage gated ion channels. Together, our data establishes that polyP could function as excitatory neuromodulator in both the PNS and CNS.

Nanomolar Bifenthrin Alters Synchronous Ca2+Oscillations and Cortical Neuron Development Independent of Sodium Channel Activity

Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca(2+) oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca(2+) indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the frequency of SCOs by 2.7-fold (EC50 = 58 nM) and decreased SCO amplitude by 36%. Changes in SCO properties were independent of modifications in voltage-gated sodium channels since 100 nM bifenthrin had no effect on the whole-cell Na(+) current, nor did it influence neuronal resting membrane potential. The L-type Ca(2+) channel blocker nifedipine failed to ameliorate bifenthrin-triggered SCO activity. By contrast, the metabotropic glutamate receptor (mGluR)5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine] normalized bifenthrin-triggered increase in SCO frequency without altering baseline SCO activity, indicating that bifenthrin amplifies mGluR5 signaling independent of Na(+) channel modification. Competitive [AP-5; (-)-2-amino-5-phosphonopentanoic acid] and noncompetitive (dizocilpine, or MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]) N-methyl-d-aspartate antagonists partially decreased both basal and bifenthrin-triggered SCO frequency increase. Bifenthrin-modified SCO rapidly enhanced the phosphorylation of cAMP response element-binding protein (CREB). Subacute (48 hours) exposure to bifenthrin commencing 2 DIV-enhanced neurite outgrowth and persistently increased SCO frequency and reduced SCO amplitude. Bifenthrin-stimulated neurite outgrowth and CREB phosphorylation were dependent on mGluR5 activity since MPEP normalized both responses. Collectively these data identify a new mechanism by which bifenthrin potently alters Ca(2+) dynamics and Ca(2+)-dependent signaling in cortical neurons that have long term impacts on activity driven neuronal plasticity.

Novel Mutations in the Extracellular Cap of the Mammalian Mechanosensitive Channel TREK-1

The Twik related potassium channel 1 (TREK-1) is one of the best studied mechanosensitive mammalian channels. TREK-1 is known to play very important roles in depression, ischemia and vasoregulation. TREK-1 belongs to the family of background or leak potassium channels are constitutively open at rest and have a central role in the tuning of neuronal resting membrane potential, duration of action potential and regulation of neurotransmitter release. These K+ channels are members of the family of K2P channels subunits containing four transmembrane and two pore domains. The functional channel is formed by two subunits and is predicted to have a two-fold symmetry.Here we show the feasibility of the use of microbial genetics to study the structure-function relationship of this mammalian channel. The advantage of this approach is that we can directly screen for channels with altered phenotypes and correlate this altered function with structural changes. We have successfully expressed a functional TREK-1 channel in bacterial cells, and show that it can partially rescue the slow growth phenotype of an E .coli strain deficient in three mayor potassium transporters. Furthermore, using random mutagenesis and bacterial screens we have isolated five mutants that better remediate the potassium deficiency of this bacterial strain. Because these mutants clustered in a stretch of 20 amino acids in the extracellular cap of the TREK-1, we think that we have found a functional hot spot by utilizing the power of bacterial genetics.

Electrophysiological Functions of Intracellular Amyloid β in Specific for Cultured Human Neurones and its Impairment Properties

Prevailing role of intracellular amyloid <TEX>${\beta}$</TEX> (<TEX>$iA{\beta}$</TEX>) in Alzheimer's disease (AD) initiation and progression attracts more and more attention in recent years. To address whether <TEX>$iA{\beta}$</TEX> induces early alterations of electrophysiological properties in cultured human primary neurons, we delivered <TEX>$iA{\beta}$</TEX> with adenovirus and measured the electrophysiological properties of infected neurons with whole-cell recordings. Our results show that <TEX>$iA{\beta}$</TEX> induces an increase in neuronal resting membrane potentials, a decrease in <TEX>$K^+$</TEX> currents and a hyperpolarizing shift in voltage-dependent activation of <TEX>$K^+$</TEX> currents. These results suggest the electrophysiological impairments induced by <TEX>$iA{\beta}$</TEX> may be responsible for its neuronal toxicity.