Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells

We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated Ca_V2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the Ca_V2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions.The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by ‘whole-cell voltage-clamp’ recordings, showing a maximum current peak in the I–V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through Ca_V2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the Ca_V2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on Ca_V2.1 calcium channels’ currents and distribution and suggest that internalization of Ca_V2.1 channels might be a mechanism of PGB action.     

Differential responses to ω-agatoxin IVA in murine frontal cortex and spinal cord derived neuronal networks

ω-Agatoxin-IVA is a well known P/Q-type Ca²⁺ channel blocker and has been shown to affect presynaptic Ca²⁺ currents as well postsynaptic potentials. P/Q-type voltage gated Ca²⁺ channels play a vital role in presynaptic neurotransmitter release and thus play a role in action potential generation. Monitoring spontaneous activity of neuronal networks on microelectrode arrays (MEAs) provides an important tool for examining this neurotoxin. Changes in extracellular action potentials are readily observed and are dependent on synaptic function. Given the efficacy of murine frontal cortex and spinal cord networks to detect neuroactive substances, we investigated the effects of ω-agatoxin on spontaneous action potential firing within these networks. We found that networks derived from spinal cord are more sensitive to the toxin than those from frontal cortex; a concentration of only 10 nM produced statistically significant effects on activity from spinal cord networks whereas 50 nM was required to alter activity in frontal cortex networks. Furthermore, the effects of the toxin on frontal cortex are more complex as unit specific responses were observed. These manifested as either a decrease or increase in action potential firing rate which could be statistically separated as unique clusters. Administration of bicuculline, a GABA_A inhibitor, isolated a single response to ω-agatoxin, which was characterized by a reduction in network activity. These data support the notion that the two clusters detected with ω-agatoxin exposure represent differential responses from excitatory and inhibitory neuronal populations.     

Camphor elicits epileptiform discharges in snail neurons: The role of ion channels modulation

Generalized convulsion is one of the most prominent manifestations of camphor toxicity, but the mechanism underlying this effect has not been elucidated. Here, we examined the excitatory and epileptogenic action of camphor in snail neurons and studied the cellular and molecular mechanisms that are involved to these effects. The spontaneous activities of neurons from subesophageal ganglia of snail Caucasotachea atrolabiata were recorded using single-electrode current clamp under control condition and after exposure to different concentrations of camphor and ion channel blockers. Under control condition, the studied neurons showed regularly spaced spontaneous action potentials. Exposure to low concentration of camphor (0.25 mM) reduced the duration of afterhyperpolarization and disrupted the spontaneous rhythmic activity, which was evidenced by an increase in the coefficient of variation of interspike intervals. The medium concentration of camphor (0.5 mM) induced more disruption in the precision of spontaneous action potential and increased the frequency of firing along with a reduction of action potential falling slope and afterhyperpolarization. Neurons showed paroxysmal depolarization shift and burst firing after exposure to camphor at high concentration (1.5 mM). We found that the blockade of K⁺ channels and upregulation of Ca²⁺ inward currents is essential for camphor-induced epileptiform activity, but the Na⁺ currents and ion channel phosphorylation with protein kinases A and C are not required. This work provided novel evidence at cellular and subcellular level that the modulation of ion channels, especially direct inhibition of K⁺ channels, is mechanistically involved to proconvulsive action of camphor.     

Anodal transcranial pulsed current stimulation: A novel technique to enhance corticospinal excitability

ObjectiveWe aimed to compare the effects of anodal-transcranial pulsed current stimulation (a-tPCS) with conventional anodal transcranial direct current stimulation (a-tDCS) on corticospinal excitability (CSE) in healthy individuals.MethodsCSE of the dominant primary motor cortex of the resting right extensor carpi radialis muscle was assessed before, immediately, 10, 20 and 30 min after application of four experimental conditions: (1) a-tDCS, (2) a-tPCS with short inter-pulse interval (a-tPCS_SIPI, 50 ms), (3) a-tPCS with long inter-pulse interval (a-tPCS_LIPI., 650 ms) and (4) sham a-tPCS. The total charges were kept constant in all experimental conditions except sham condition. The outcome measure in this study was motor evoked potentials.ResultsOnly a-tDCS and a-tPCS_SIPI (P < 0.05) induced significant increases in CSE, lasted for at least 30 min. Post-hoc tests indicated that this increase was larger in a-tPCS_SIPI (P < 0.05). There were no significant changes following application of a-tPCS_LIPI and sham a-tPCS. All participants tolerated the applied currents in all experimental conditions very well.ConclusionsCompared to a-tDCS, a-tPCS_SIPI is a better technique for enhancement of CSE. There were no sham effects for application of a-tPCS. However, unlike a-tDCS which modifies neuronal excitability by tonic depolarization of the resting membrane potential, a-tPCS modifies neuronal excitability by a combination of tonic and phasic effects.Significancea-tPCS could be considered as a promising neuromodulatory tool in basic neuroscience and as a therapeutic technique in neurorehabilitation.     

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75 antagonists

Two-pore potassium (K_2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gα_i-coupled hP2Y₁₂ receptor, ADP and ATP significantly inhibited hK_2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y₁₂ antagonist AR-C69931MX, the hP2Y₁ antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gα_q/11-coupled hP2Y₁ receptor, ADP and ATP also inhibited hK_2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K_2P2.1 channels, ADP-induced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gα_q/11- and Gα_i-coupled P2Y receptors inhibit K_2P channels and the action of hP2Y₁₂ receptors appears to involve co-activation of endogenous hP2Y₁ receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity.     

Aberrant modulation of a delayed rectifier potassium channel by glutamate in Alzheimer's disease

In Alzheimer's disease (AD), potassium channel abnormalities have been reported in both neural and peripheral tissues. Herein, using whole-cell patch-clamp, we demonstrate an aberrant glutamate-dependent modulation of K_V1.3 channels in T lymphocytes of AD patients. Although intrinsic K_V1.3 properties in patients were similar to healthy individuals, glutamate (1–1000 μM) failed to yield the hyperpolarizing shift normally observed in K_V1.3 steady-state inactivation (? 4.4 ± 2.7 mV in AD vs. ? 14.3 ± 2.5 mV in controls, 10 μM glutamate), resulting in a 4-fold increase of resting channel activity. Specific agonist and antagonist data indicate that this abnormality is due to dysfunction of cognate group II mGluRs. Given that glutamate is present in plasma and that both mGluRs and K_V1.3 channels regulate T-lymphocyte responsiveness, our finding may account for the presence of immune-associated alterations in AD. Furthermore, if this aberration reflects a corresponding one in neural tissue, it could provide a potential target in AD pathogenesis.     

Suppression of ATP-induced excitability in rat small-diameter trigeminal ganglion neurons by activation of GABAB receptor

The aim of the present study was to investigate whether a GABA_B receptor agonist could modulate ATP-activated neuronal excitability of nociceptive TRG neurons using perforated whole-cell patch-clamp and immunohistochemical techniques. Immunohistochemical analysis revealed that 86% of P₂X₃ receptor-immunoreactive, small-diameter TRG neurons co-expressed GABA_B receptor. Under voltage-clamp conditions (V_h = −60 mV), application of ATP activated the inward current in acutely isolated rat TRG neurons in a dose-dependent manner (10–50 μM) and this current could be blocked by pyridoxal-phosphate-6-azophenyl-27,47-disulfonic acid (PPADS) (10 μM), a selective P₂ purinoreceptor antagonist. The peak amplitude of ATP-activated currents was significantly inhibited after application of GABA_B receptor agonist, baclofen (10–50 μM), in a concentration-dependent and reversible manner. The baclofen-induced inhibition of ATP-activated current was abolished by co-application of 3-amino-2 (4-chlorophenyl)-2hydroxypropysufonic acid) saclofen, a GABA_B receptor antagonist (50 μM). Under current-clamp conditions, application of 20 μM ATP significantly depolarized the membrane potential resulting in increased mean action potential frequencies, and these ATP-induced effects were significantly inhibited by baclofen and these effects were antagonized by co-application of saclofen. Together, the results suggested that GABA_B receptor activation could inhibit the ATP-induced excitability of small-diameter TRG neurons activated through the P₂X₃ receptor. Thus, the interaction between P₂X₃ and GABA_B receptors of small-diameter TRG neuronal cell bodies is a potential therapeutic target for the treatment of trigeminal nociception.     

Two mechanisms underlie the slow noradrenergic depolarization in the rat tail artery in vitro

In rat tail artery, short trains of electrical stimuli evoke both ATP-mediated excitatory junction potentials (EJPs) and a slow noradrenaline (NA)-mediated depolarization (NAD). Here we have investigated the contribution of α₁- and α₂-adrenoceptors to the NAD. The α₁-adrenoceptor antagonist, prazosin (0.1 μM), and the α₂-antagonist, rauwolscine (1 μM), reduced the amplitude of the NAD and in combination these agents virtually abolished the NAD. The K_ATP channel blocker, glibenclamide (10 μM) abolished the α₂-adrenoceptor-mediated component of the NAD, indicating that activation of these receptors produces closure of K_ATP channels. The α₁-adrenoceptor-mediated component of the NAD was increased in amplitude by glibenclamide. Changes in membrane conductance were monitored by measuring the time constant of decay of EJPs (τEJP). The τEJP was increased during α₁-adrenoceptor-mediated depolarization, indicating a decrease in membrane conductance; i.e. closure of K⁺ channels. Broad-spectrum K⁺ channel blockers (tetraethylammonium, 4-aminopyridine, Ba²⁺) and the TASK-1 K⁺ channel blocker, anandamide (10 μM), did not reduce the α₁-adrenoceptor-mediated NAD. The α₁-adrenoceptor-mediated NAD was unaffected by the Cl^? channel blockers, 9-anthracene carboxylic acid (100 μM) and niflumic acid (10 μM) or by the non-selective cation channel blocker, SKF 96365 (10 μM). These findings indicate that the NAD is produced by activation of both α₁-and α₂-adrenoceptors. The α₂-adrenoceptor-mediated component is produced by closure of K_ATP channels whereas the α₁-adrenoceptor-mediated component is most likely mediated by closure of another type of K⁺ channel.     

Regulation of voltage-gated Ca2 + currents by Ca2 +/calmodulin-dependent protein kinase II in resting sensory neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca^2 + channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca^2 + currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0 μM) reduced depolarization-induced I_Ca by 16–30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total I_Ca. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.     

Assessing the effects of the neonicotinoid insecticide imidacloprid in the cholinergic synapses of the stellate cells of the mouse cochlear nucleus using whole-cell patch-clamp recording

Imidacloprid (IMI) is widely used systemic insecticide that acts as an agonist on nicotinic acetylcholine receptors (nAChRs). IMI has been reported to be more active against insect nAChRs (EC₅₀ 0.86–1 μM) than it is against mammalian nAChRs (EC₅₀ 70 μM). The objective of this study was to determine to what extent IMI affects the nAChRs of the stellate cells of mouse cochlear nucleus (CN), using whole-cell patch-clamp recording. Puff application of 1 μM IMI had no significant effect on the membrane properties of the neurons tested, while a concentration of 10 μM caused a significant depolarizing shift in the membrane potential and resulted in increases in the fluctuation of the membrane potential and in the frequency of miniature postsynaptic potentials (mpps) within less than a minute of exposure. IMI at concentrations ≥50 μM caused a significant depolarizing shift in the membrane potential, accompanied by a marked increase in the frequency of action potential. IMI decreased the membrane input resistance and the membrane time constants. Bath application of 50 μM d-tubocurarine (d-TC) reversibly blocked the depolarizing shift of the resting membrane potential and the spontaneous firing induced by IMI application in current clamp and blocked the inward currents through nicotinic receptors induced by IMI application in voltage clamp. Similarly, 100 nM α-bungarotoxin (α-BgTx) blocked the spontaneous firing induced by IMI (n = 3). The amplitude of the 100 μM IMI-induced inward current at ?60 mV holding potential was 115.0 ± 16.2 pA (n = 7). IMI at a concentration of 10 μM produced 11.3 ± 3.4 pA inward current (n = 4). We conclude that exposure to IMI at concentrations ≥10 μM for <1 min can change the membrane properties of neurons that have nAChRs and, as a consequence, their function.     

Arsenic induced neuronal apoptosis in guinea pigs is Ca2+ dependent and abrogated by chelation therapy: Role of voltage gated calcium channels

Arsenic contaminated drinking water has affected more than 200 million people globally. Chronic arsenicism has also been associated with numerous neurological diseases. One of the prime mechanisms postulated for arsenic toxicity is reactive oxygen species (ROS) mediated oxidative stress. In this study, we explored the kinetic relationship of ROS with calcium and attempted to dissect the calcium ion channels responsible for calcium imbalance after arsenic exposure. We also explored if mono- or combinational chelation therapy prevents arsenic-induced (25 ppm in drinking water for 4 months) neuronal apoptosis in a guinea pig animal model. Results indicate that chronic arsenic exposure caused a significant increase in ROS followed by NO and calcium influx. This calcium influx is mainly dependent on L-type voltage gated channels that disrupt mitochondrial membrane potential, increase bax/bcl₂ levels and caspase 3 activity leading to apoptosis. Interestingly, blocking of ROS could completely reduce calcium influx whereas calcium blockage partially reduced ROS increase. While in general mono- and combinational chelation therapies were effective in reversing arsenic induced alteration, combinational therapy of DMSA and MiADMSA was most effective. Our results provide evidence for the role of L-type calcium channels in regulating arsenic-induced calcium influx and DMSA + MiADMSA combinational therapy may be a better protocol than monotherapy in mitigating chronic arsenicosis.     

Generation of hydrogen peroxide mediates hanging death-induced neuronal cell apoptosis in the dentate gyrus of the rat brain

Present study was aimed to find out whether hanging death (HD) induces generation of reactive oxygen species (ROS) and neuronal cell apoptosis in the dentate gyrus (DG) region of rat brain. Permanent global brain ischemia was generated by HD in experimental rats and the brain was isolated after 0, 1, 2, 3, 4, 5, 6, 9, 12 and 24 h post- HD and cervical dislocation (CD). The histology, hydrogen peroxide (H₂O₂) concentration, catalase, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed in neuronal cells of DG region of the brain. Permanent global brain ischemia generated due to HD induced generation of H₂O₂ as well as catalase activity. The increased level of H₂O₂ was associated with the increased caspase-9 and caspase-3 activities. The increased caspase-3 activity induced neuronal cell apoptosis during early period (0–9 h) of HD as compare to CD group. The neuronal cells necrosis was observed only 12 h post-HD, while CD induced necrosis as early as 3 h post-CD and the histoarchitecture of DG region was gradually disrupted after 6 h of CD. In conclusion, data of the present study suggest that the permanent global brain ischemia induces neuronal cell apoptosis during early period of HD through ROS-mediated pathway, while CD induces neuronal cell necrosis and disruption of the histoarchitecture of the DG region. Thus, neuronal cell apoptosis may be used to develop a cellular marker to find out the exact timing of HD.     

Molecular and cellular influences of permethrin on mammalian nociceptors at physiological temperatures

The influence of pyrethroid insecticides is thought to be abrogated at mammalian physiological temperatures. Yet there are many reports of transient pain and paresthesia following accidental exposures. Using whole cell patch clamp techniques, we examined the interaction of the pyrethroid insecticide permethrin on skin, muscle and putative vascular nociceptors of the rat DRG (dorsal root ganglion). Following permethrin (10 μM) application, action potential (AP) duration was increased in all nociceptor populations, but only muscle nociceptors developed spontaneous activity or increased excitability (tests at 21 °C). TTX (tetrodotoxin) did not prevent the development of spontaneous activity or reduce excitability. We examined the influence of permethrin on TTX resistant channel proteins that control excitability and spontaneous activity (Na_v1.8, voltage-gated sodium channel 1.8; K_v7, voltage gated potassium channel 7). In all nociceptor populations, permethrin increased the tau of deactivation (taudeact), in a voltage dependent manner, and hyperpolarized the V_1/2 for activation over 10 mV. There were no permethrin dependent influences on K_v7, or on the voltage dependence of inactivation of Na_v1.8. The influence of permethrin on AP duration, after hyperpolarization, spontaneous activity, half-activation potential (V_1/2) and taudeact were reduced, but not fully reversed, when tests were conducted at 35 °C. In conclusion, permethrin greatly modifies the voltage dependent activation and deactivation of Na_v1.8 expressed in skin, muscle and vascular nociceptors. These influences remain significant at 35 °C. One population of muscle nociceptors exhibited a unique vulnerability to the acute administration of permethrin manifested as increased excitability and spontaneous activity.     

Pharmacological characterization of histamine-gated chloride channels from the housefly Musca domestica

The biogenic amine histamine (HA) is not only the neurotransmitter of photoreceptors but also has important roles in mechanosensory reception, temperature preference, sleep and olfactory processing in insects. Two cDNAs (MdhclA and MdhclB) that encode HA-gated chloride channel subunits (MdHCLA and MdHCLB) were cloned from the housefly Musca domestica. The cRNAs were injected into Xenopus laevis oocytes to examine the functions and pharmacological characteristics of MdHCLA and MdHCLB channels using a two-electrode voltage clamp method. HA was used to activate MdHCLA and MdHCLB channels to evoke inward currents with EC₅₀s of 33.1 μM and 6.28 μM, respectively. 2-(3-Trifluoromethylphenyl)histamine, an HA H₁ receptor agonist, was a partial agonist of MdHCLB receptors with an EC₅₀ of 49.4 μM. MdHCLB channels were also activated by γ-aminobutyric acid (GABA) and monoamines, such as octopamine, serotonin (5-HT) and dopamine (DA); 5-HT and DA also acted as competitive antagonists. GABA acted as a full agonist of MdHCLB receptors with an EC₅₀ of 1.11 mM. d-Tubocurarine, cimetidine and picrotoxinin were poor inhibitors of HA- and GABA-evoked currents in MdHCLB channels. Our data show that HCLB channels are more sensitive to agonists when compared with HCLA channels. HCLB channels are also affected by antagonists but insusceptible to known insecticides that target GABA- and glutamate-gated chloride channels.     

An electrophysiological study on the effects of BDNF and FGF2 on voltage dependent Ca2 + currents in developing human striatal primordium

Over the past decades, studies in both Huntington's disease animal models and pilot clinical trials have demonstrated that replacement of degenerated striatum and repair of circuitries by grafting fetal striatal primordium is feasible, safe and may counteract disease progression. However, a better comprehension of striatal ontogenesis is required to assess the fetal graft regenerative potential. During neuronal development, neurotrophins exert pleiotropic actions in regulating cell fate and synaptic plasticity. In this regard, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are crucially implicated in the control of fate choice of striatal progenitor cells. In this study, we intended to refine the functional features of human striatal precursor (HSP) cells isolated from ganglionic eminence of 9–12 week old human fetuses, by studying with electrophysiological methods the effect of BDNF and FGF2 on the membrane biophysical properties and the voltage-dependent Ca^2 + currents. These features are particularly relevant to evaluate neuronal cell functioning and can be considered reliable markers of the developmental phenotype of human striatal primordium. Our results have demonstrated that BDNF and FGF2 induced membrane hyperpolarization, increased the membrane capacitance and reduced the resting total and specific conductance values, suggesting a more efficient control of resting ionic fluxes. Moreover, the treatment with both neurotrophins enhanced N-type Ca^2 + current amplitude and reduced L- and T-type ones. Overall, our data indicate that BDNF and FGF2 may help HSP cells to attain a more functionally mature phenotype.     

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca^2 + channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca^2 + channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca²⁺ currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca^2 + current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca²⁺ channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Ca_v1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca²⁺ channels. Moreover, pharmacological inactivation of L-type Ca^2 + channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca^2 + channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Ca_v1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

Angiotensin II increases excitability and inhibits a transient potassium current in vagal primary sensory neurons

The octapeptide angiotensin II (ANG II) plays a pivotal role in the maintenance of blood pressure by activating ANG II receptors located in variety of cell types including neurons housed in the central nervous system (CNS) and in the peripheral nervous system (PNS). ANG II (100 nM) blocked spike frequency accommodation (SFA) recorded with whole-cell patch technique in acutely isolated nodose ganglion neurons (NGN) from adult rats. ANG II increased the frequency of action potentials (AP) produced by supramaximal 500 ms depolarizing currents recorded in both tonic (16 Hz vs. 58 Hz, control vs. ANG II perfusion respectively, n = 9) and phasic (1 Hz vs. 38 Hz, n = 13) NGNs. ANG II produced no significant changes in: the resting membrane potential (?51 mV vs. ?50 mV, n = 65), AP overshoot (46 mV vs. 41 mV, n = 25), AP undershoot (?65 mV vs. ?61 mV, n = 25), AP duration (1 ms vs. 1.2 ms, n = 25), and AP threshold (?40 mV vs. ?43 mV, n = 19). CV-11974 (600 nM), a specific AT1 receptor antagonist, prevented ANG II-evoked changes SFA (n = 10).ANG II (100 nM) had no significant effect on total outward potassium current (I_K) but inhibited a fast activating and fast inactivating I_K recorded in the presence of TEA. A kinetically similar I_K was also inhibited by 4-AP (3 mM). In phasic NGNs, 4-AP occluded the effects of 100 nM ANG II on SFA. Our results indicate that ANG II can block an A-type of I_K and that this effect may underlie the ANG II-mediated change in SFA.     

Transforming growth factor‐β1 primes proliferating adult neural progenitor cells to electrophysiological functionality

The differentiation of adult neural progenitors (NPCs) into functional neurons is still a limiting factor in the neural stem cell field but mandatory for the potential use of NPCs in therapeutic approaches. Neuronal function requires the appropriate electrophysiological properties. Here, we demonstrate that priming of NPCs using transforming growth factor (TGF)‐β1 under conditions that usually favor NPCs' proliferation induces electrophysiological neuronal properties in adult NPCs. Gene chip array analyses revealed upregulation of voltage‐dependent ion channel subunits (Kcnd3, Scn1b, Cacng4, and Accn1), neurotransmitters, and synaptic proteins (Cadps, Snap25, Grik4, Gria3, Syngr3, and Gria4) as well as other neuronal proteins (doublecortin [DCX], Nrxn1, Sept8, and Als2cr3). Patch‐clamp analysis demonstrated that control‐treated cells expressed only voltage‐dependent K⁺‐channels of the delayed‐rectifier type and the A‐type channels. TGF‐β1‐treated cells possessed more negative resting potentials than nontreated cells owing to the presence of delayed‐rectifier and inward‐rectifier channels. Furthermore, TGF‐β1‐treated cells expressed voltage‐dependent, TTX‐sensitive Na⁺ channels, which showed increasing current density with TGF‐β1 treatment duration and voltage‐dependent (+)BayK8644‐sensitive L‐Type Ca²⁺ channels. In contrast to nontreated cells, TGF‐β1‐treated cells responded to current injections with action‐potentials in the current‐clamp mode. Furthermore, TGF‐β1‐treated cells responded to application of GABA with an increase in membrane conductance and showed spontaneous synaptic currents that were blocked by the GABA‐receptor antagonist picrotoxine. Only NPCs, which were treated with TGF‐β1, showed Na⁺ channel currents, action potentials, and GABAergic currents. In summary, stimulation of NPCs by TGF‐β1 fosters a functional neuronal phenotype, which will be of relevance for future cell replacement strategies in neurodegenerative diseases or acute CNS lesions. GLIA 2013;61:1767–1783     

Propofol facilitated excitatory postsynaptic currents frequency on nucleus tractus solitarii (NTS) neurons

Propofol, an intravenous anesthetic, is broadly used for general anesthesia and diagnostic sedations due to its fast onset and recovery. Propofol depresses respiratory and cardiovascular reflex responses, however, their underlying mechanisms are not well known. Cardiorespiratory information from visceral afferent vagus nerves is integrated in the nucleus tractus solitarii (NTS). Cardiac and respiratory signals transducing vagal afferent neurons release the excitatory neurotransmitter glutamate onto NTS neurons in an activity dependent manner and trigger negative feedback reflex responses. In this experiment, the effects of propofol on glutamatergic synaptic responses at NTS neurons was tested using patch clamp methods. Glutamatergic excitatory postsynaptic currents (EPSC) were recorded at chloride reversal potential (? 49 mV) without γ-aminobutyric acid type A (GABA_A) receptor antagonists. Propofol (≥ 3 μM) facilitated frequency of the spontaneous EPSCs in a concentration dependent manner without altering amplitude and decay time. The GABA_A receptor selective antagonist, gabazine (6 μM), attenuated propofol effects on glutamate release. Propofol (10 μM) evoked glutamate release was also blocked in the presence of the voltage dependent Na⁺ and Ca²⁺ channel blockers TTX (0.3 μM) and Cd²⁺ (0.2 mM), respectively. In addition, the Na⁺–K⁺–Cl^? cotransporter type 1 antagonist bumetanide (10 μM) also inhibited propofol evoked increase in sEPSC frequency. These results suggest that propofol evoked glutamate release onto NTS neurons by GABA_A receptor-mediated depolarization of the presynaptic excitatory terminals.