Abstract Introduction A widely debated function of sleep involves a homeostatic program of down-regulation of excitatory synaptic strength following an overall increase during the preceding waking period, preserving however the previously existing synaptic weights associated with newly acquired memories. We tested this hypothesis by applying thorough statistical analysis of parameters of excitatory and inhibitory miniature postsynaptic currents (mEPSC/mIPSC) recorded ex vivo in mouse cortical pyramidal neurons at three characteristic wake/sleep stages. Methods Cingulate cortex coronal slices were obtained at fixed Zeitgeber time (ZT6, to control for circadian clock) from control C57BL6 (MEF2C f/f) mice subjected to 6h acute sleep deprivation (SD), recovery sleep =4hSD+2h(RS), or 6h control sleep (CS). mEPSCs and mIPSCs were recorded from functionally identified whole-cell patch-clamped pyramidal neurons in cortical layer 2/3 (L2/3). Statistical analysis of frequencies, amplitudes, and charge transfer rates of mEPSCs and mIPSCs was done using non-parametric Kruskal-Wallis multiple comparison test and K-means clustering test. Results mEPSC frequency (F) and charge transfer (CT) were significantly reduced for RS and CS compared to SD (F: -57%, -47%; CT: -64%, -55%). mEPSC amplitude (A) was significantly reduced for CS compared to SD (-15%). Two-centroid clustering test revealed that analyzed parameters of F, A and CT for SD condition were approximately evenly split between upper and lower range clusters, while the same parameters for RS and CS conditions revealed a pronounced redistribution (>75% lower-, <25% upper ranges). Wake/sleep state related changes of mIPSC parameters showed opposite pattern compared to excitatory synapses. All three parameters were increased in RS vs. SD (F: +63%, A: +7%, CT: +42%) and this difference reached significance levels in CS vs. SD (F: +88%, A: +24%, CT: +109%). Clustering analysis of mIPSC parameters revealed mostly stable distribution pattern between upper and lower ranges for all wake/sleep states. Conclusion Significant changes in excitatory/inhibitory balance in the frontal cortex is part of the homeostatic response upon transition from wakefulness to various phases of sleep. The excitatory component prevails during wakefulness, while the inhibitory component peaks during sleep. Support (If Any) NIH grant R01 NS103422 to RWG.

Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.

Neuronal activity and gene expression in response to the loss of sleep can provide a window into the enigma of sleep function. Sleep loss is associated with brain differential gene expression, an increase in pyramidal cell mEPSC frequency and amplitude, and a characteristic rebound and resolution of slow wave sleep-slow wave activity (SWS-SWA). However, the molecular mechanism(s) mediating the sleep-loss response are not well understood. We show that sleep-loss regulates MEF2C phosphorylation, a key mechanism regulating MEF2C transcriptional activity, and that MEF2C function in postnatal excitatory forebrain neurons is required for the biological events in response to sleep loss in C57BL/6J mice. These include altered gene expression, the increase and recovery of synaptic strength, and the rebound and resolution of SWS-SWA, which implicate MEF2C as an essential regulator of sleep function.

Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K+. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K+ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K+, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na+ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na+ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm2 or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na+ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na+ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of NaV, CaV, and KV channels associated with human disease.

Regulation of organellar fusion and fission by Ca2+ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca2+-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca2+ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca2+ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca(2+) from the acidic endolysosomes by activation of the two-pore channels TPC1 and TPC2. The channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers. The native and recombinant TPC1 channels are activated by NAADP. TPC1 activity requires acidic luminal pH and high luminal Ca(2+). With Ba(2+) as the permeable ion, luminal Ca(2+) activates TPC1 with an apparent K(m) of 180 μm. TPC1 operates in two tightly coupled conductance states of 47 ± 8 and 200 ± 9 picosiemens. Importantly, opening of the large conductance markedly increases the small conductance mean open time. Changes in membrane potential from 0 to -60 mV increased linearly both the small and the large conductances and NP(o), indicating that TPC1 is regulated by voltage. Intriguingly, the apparent affinity for activation of TPC1 by its ligand NAADP is not constant. Rather, hyperpolarization increases the apparent affinity of TPC1 for NAADP by 10 nm/mV. The concerted regulation of TPC1 activity by luminal Ca(2+) and by membrane potential thus provides a potential mechanism to explain NAADP-induced Ca(2+) oscillations. These findings reveal unique properties of TPC1 to explain its role in Ca(2+) oscillations and cell function.

Background: Evolutionary conserved Bax inhibitor-1 (BI-1) protects against ER stress-mediated apoptosis. Results: We identified a Ca2+-permeable channel pore in the C terminus of BI-1. Critical pore properties are an α-helical structure and two aspartate residues conserved among animals, but not among plants and yeast. Conclusion: C-terminal domain of BI-1 harbors a Ca2+-permeable channel pore. Significance: BI-1 has Ca2+ channel properties likely relevant for its function in ER stress and apoptosis. Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca2+ homeostasis by acting as a Ca2+-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca2+ pore responsible for its Ca2+-leak properties. We utilized a set of C-terminal peptides to screen for Ca2+ leak activity in unidirectional 45Ca2+-flux experiments and identified an α-helical 20-amino acid peptide causing Ca2+ leak from the ER. The Ca2+ leak was independent of endogenous ER Ca2+-release channels or other Ca2+-leak mechanisms, namely translocons and presenilins. The Ca2+-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca2+-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca2+-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca2+-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca2+ leak. Thus, we elucidated residues critically important for BI-1-mediated Ca2+ leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca2+-leak properties of BI-1 are an added function during evolution.

PURPOSE:Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca(2+) homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca(2+) concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca(2+) release channels, transient receptor potential polycystin family (TRPP) channels.METHODS:Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca(2+) channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system.RESULTS:All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues.CONCLUSIONS:The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology.

BACKGROUND:Hypokalemic periodic paralysis (HypoPP) is associated with mutations in either the Ca(V)1.1 calcium channel or the Na(V)1.4 sodium channel. Some Na(V)1.4 HypoPP mutations have been shown to cause an anomalous inward current that may contribute to the attacks of paralysis. Herein, we test whether disease-associated Na(V)1.4 mutations in previously untested homologous regions of the channel also give rise to the anomalous current.METHODS:The functional properties of mutant Na(V)1.4 channels were studied with voltage-clamp techniques in an oocyte expression system.RESULTS:The HypoPP mutation Na(V)1.4-R1132Q conducts an anomalous gating pore current, but the homologous R1448C mutation in paramyotonia congenita does not.CONCLUSIONS:Gating pore currents arising from missense mutations at arginine residues in the voltage sensor domains of Na(V)1.4 are a common feature of HypoPP mutant channels and contribute to the attacks of paralysis.

Ca(2+) release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca(2+)-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical "nongenomic" effects mediated by estrogen receptors (ER) include rapid Ca(2+) release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of colocalization between RyR type 2 (RyR2) and ER type beta (ER beta) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single-channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ER beta (ER beta 1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca(2+)] concentrations of 100 nM, suggesting a synergistic action of ER beta 1 and Ca(2+) in RyR activation, and a potential contribution to Ca(2+)-induced Ca(2+) release rather than to basal intracellular Ca(2+) concentration level at rest. This RyR/ER beta interaction has potential effects on cellular physiology, including roles of shorter ER beta isoforms and modulation of the RyR/ER beta complexes by exogenous estrogens.

The outer hair cell from Corti's organ possesses voltage-dependent intramembranous molecular motors evolved from the SLC26 anion transporter family. The motor, identified as prestin (SLC26a5), is responsible for electromotility of outer hair cells and mammalian cochlear amplification, a process that heightens our auditory responsiveness. Here, we describe experiments designed to evaluate the effects of anions on the motor's voltage-sensor charge movement, focusing on prestin's voltage-dependent Boltzmann characteristics. We find that the nature of the anion, including species, valence, and structure, regulates characteristics of the charge movement, signifying that anions play a more complicated role than simple voltage sensing in cochlear amplification.

Ryanodine receptors (RyRs) amplify intracellular Ca2+ signals by massively releasing Ca2+ from intracellular stores. Exaggerated chronic Ca2+ release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca2+-dependent apoptosis, providing a basis for the “calcium hypothesis” of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF1–82), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF1–82 strongly increased both mean currents (EC50=12nM, Hill coefficient (nH)∼1) and open probability for higher sublevels for single RyR channels (EC50=7nM, nH∼2). Bell-shaped Ca2+-activation curve remained unchanged, suggesting that PS1 NTF1–82 allosterically potentiates RyRs, but that the channel still requires Ca2+ for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF1–82 was overridden by receptor desensitization at high [Ca2+] (pCa>5). This potentiation of RyR by PS1 NTF1–82 reveals a new mechanism of physiologically relevant PS1-regulated Ca2+ release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca2+ leak channels formed by PS1 holoproteins.

Presenilin-1 (PS1) and presenilin-2 (PS2) form the catalytic core in γ-secretase complexes and mutations in these proteins result in aberrant cleavage of amyloid precursor protein leading to accumulation of the β-amyloid in the brain of familial Alzheimer Disease patients. PS2 possesses a hydrophilic cytoplasmic N-terminal domain (PS2 NTF1-87) dispensable for γ-secretase activity with physiological functions yet to be determined. The effects of this soluble 87 amino acid fragment of mouse PS2 on single channel activity of mouse brain ryanodine receptors (RyR) were determined. PS2 NTF1-87 application to the cytoplasmic side of the RyR significantly increased single channel activity by favoring higher sublevel openings. The Ca2+ activation and desensitization ranges for RyRs were unchanged. We demonstrate facilitation of RyR gating by PS2 NTF1-87, which might represent a general mechanism of RyR regulation by presenilins potentially prone to be affected by mutations or external stimuli contributing to the development of neurodegenerative diseases.

Recently, we identified an outer hair cell (OHC) lateral membrane conductance, G metL, that colocalizes with prestin and passes Cl−, thereby influencing prestin’s (SLC26A5) electromechanical activity. In this study, we report on a comparison of the temperature and tension dependence of G metL and prestin. Though we find significant temperature and tension dependence of each, substantial differences exist which indicate their independent identity. The following data support this conclusion: (1) The voltage dependence of G metL does not follow that of prestin’s nonlinear capacitance (NLC) function when the latter is shifted by either temperature or membrane tension; (2) Unlike native OHCs whose NLC can be modulated by influx of extracellular Cl−, prestin-transfected Chinese hamster ovary (CHO) cells do not show this phenomenon; (3) Stretch-sensitive, single channel currents are not evidenced after prestin transfection in CHO cells; and (4) There is no correlation between prestin expression level (gauged via NLC) and transmembrane current through G metL. Thus, G metL must result from the activity of another molecular species within the lateral membrane of the OHC. A clue to its identity is the conductance’s nonlinear temperature dependence in contrast to prestin and other OHC conductances’ linear dependence. Whereas K+ conductances in OHCs present a uniform Q 10 close to 1.2, G metL shows a bimodal Q 10, with a Q 10 of 1.5 below 34°C and a Q 10 of greater than 4 and above. The dissociation of SLC26A5 (prestin) and G metL theoretically provides for a modifiable anionic feedback to prestin via the degree of spatial separation between these interacting partners within the OHC lateral membrane.

A variety of ionic channels control cationic content of outer hair cells (OHC). However, little is known about mechanisms that control intracellular anions. In this work we demonstrate that monovalent anions (Cl-) and cations (K+, Na+) can permeate the OHC membrane through a voltage dependent, anion/cation non-selective pathway. Moreover, Cl- flux through these putative channels sufficiently modulates intracellular Cl- concentration to influence the behavior of the OHC membrane motor protein prestin. Depletion of OHC intracellular and extracellular Cl- ions does not eliminate the OHC's nonlinear capacitance that derives from the molecule's mobile charge. That is, total charge, Q(max), was not reduced upon intracellular and extracellular Cl- substitution by HEPES for prolonged periods of time. Additionally, following intracellular Cl- substitution by the divalent anion sulfate, the apparent valence of prestin's mobile charge was stable (0.7-0.8) and Q(max) remained substantial. Our data argue against the hypothesis that Cl- is an exogenous voltage sensor for prestin.

Outer hair cells underlie high frequency cochlear amplification in mammals. Fast somatic motility can be driven by voltage-dependent conformational changes in the motor protein, prestin, which resides exclusively within lateral plasma membrane of the cell. Yet, how a voltage-driven motor could contribute to high frequency amplification, despite the low-pass membrane filter of the cell, remains an enigma. The recent identification of prestin's Cl- sensitivity revealed an alternative mechanism in which intracellular Cl- fluctuations near prestin could influence the motor. We report the existence of a stretch-sensitive conductance within the lateral membrane that passes anions and cations and is gated at acoustic rates. The resultant intracellular Cl- oscillations near prestin may drive motor protein transitions, as evidenced by pronounced shifts in prestin's state-probability function along the voltage axis. The sensitivity of prestin's state probability to intracellular Cl- levels betokens a more complicated role for Cl- than a simple extrinsic voltage sensor. Instead, we suggest an allosteric modulation of prestin by Cl- and other anions. Finally, we hypothesize that prestin sensitivity to anion flux through the mechanically activated lateral membrane can provide a driving force that circumvents the membrane's low-pass filter, thus permitting amplification at high acoustic frequencies.

The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K+ currents (I(K)) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type) inactivation. With 2 mM [Mg(2+)](o), I(K) activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC(50) = 11 microM) rapidly (12 s) inhibited I(K) in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 microM) did not influence whole-cell I(K), although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 microM) had no influence on I(K). Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I(K) were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 +/- 0.5 to 3.2 +/- 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 microM) reduced an open-channel probability (from 0.45 +/- 0.1 in control to 0.1 +/- 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABAA receptor function. Etifoxine displaced [35S]TBPS (t-butylbicyclophosphorothionate) from GABAA receptors of rat cortical membranes with an IC50 of 6.7±0.8 μM and [3H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC50 of 27.3±1.0 μM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABAA receptor-mediated membrane currents elicited by submaximal (5–10 μM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 μM which reflected the post-synaptic potentiation of a small (∼20 pA) tonic and bicuculline-sensitive GABAA receptor-gated Cl− current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 μM), an antagonist of central-type benzodiazepine sites present at GABAA receptors, but were partly inhibited by PK11195 (10 μM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABAA receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

Transmission and processing of nociceptive information in the superficial dorsal horn (DH) of the spinal cord involves activation of AMPA-type glutamate receptors (AMPARs). We have studied the properties of native AMPARs in freshly dissociated laminae I-II neurones from postnatal rats using a modified form of the concentration-clamp technique for fast agonist application. Analysis of kainic acid dose-response curves showed the existence of two types of functional agonist binding sites governing AMPAR activation. These sites differ by their affinity for the agonist. Depending on the neurotransmitter concentration reached in the synaptic cleft, the relative contribution of high affinity and low affinity sites might play an important role in the shaping of AMPAR-mediated postsynaptic currents.