Channels In Rat Hippocampal Neurons Research Articles

The effects of gastrin-releasing peptide on the voltage-gated channels in rat hippocampal neurons

Gastrin-releasing peptide (GRP) has been implicated in several aspects of physiology and behavior including digestion, cancer, lung development, and memory process. Increasing evidence in rodents shows that GRP may contribute to hippocampal circuit function. Though the central role of GRP in the brain has been established, the cellular and molecular mechanisms of its actions have not been well defined. Thus in this study, we verified the expression of GRPR in the rat hippocampal CA1 region. Then we examined the mechanisms closely related to neuronal excitability, the effects of GRP on voltage-gated ion channels in CA1 neurons using patch-clamp. The results showed that GRP could decrease voltage-gated sodium currents mainly by affecting the kinetics of recovery from the inactivated state. However, GRP enhanced both kinds of voltage-gated potassium channels, the A-type channels were more sensitive to GRP than K-type channels. In conclusion, we found that GRP could alter the voltage-gated Na+ and K+ ion channel characteristics which might be the ionic mechanisms of the physiological function of GRP in the brain.

Antiepileptic geissoschizine methyl ether is an inhibitor of multiple neuronal channels.

Geissoschizine methyl ether (GM) is an indole alkaloid isolated from Uncaria rhynchophyll (UR) that has been used for the treatment of epilepsy in traditional Chinese medicine. An early study in a glutamate-induced mouse seizure model demonstrated that GM was one of the active ingredients of UR. In this study, electrophysiological technique was used to explore the mechanism underlying the antiepileptic activity of GM. We first showed that GM (1−30 μmol/L) dose-dependently suppressed the spontaneous firing and prolonged the action potential duration in cultured mouse and rat hippocampal neurons. Given the pivotal roles of ion channels in regulating neuronal excitability, we then examined the effects of GM on both voltage-gated and ligand-gated channels in rat hippocampal neurons. We found that GM is an inhibitor of multiple neuronal channels: GM potently inhibited the voltage-gated sodium (NaV), calcium (CaV), and delayed rectifier potassium (IK) currents, and the ligand-gated nicotinic acetylcholine (nACh) currents with IC50 values in the range of 1.3−13.3 μmol/L. In contrast, GM had little effect on the voltage-gated transient outward potassium currents (IA) and four types of ligand-gated channels (γ-amino butyric acid (GABA), N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainite (AMPA/KA receptors)). The in vivo antiepileptic activity of GM was validated in two electricity-induced seizure models. In the maximal electroshock (MES)-induced mouse seizure model, oral administration of GM (50−100 mg/kg) dose-dependently suppressed generalized tonic-clonic seizures. In 6-Hz-induced mouse seizure model, oral administration of GM (100 mg/kg) reduced treatment-resistant seizures. Thus, we conclude that GM is a promising antiepileptic candidate that inhibits multiple neuronal channels.

Quantitative determination of talatisamine and its pharmacokinetics and bioavailability in mouse plasma by UPLC-MS/MS

Talatisamine, as the efficacy ingredient of Aconitum, was known as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 μm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1–1000 ng/mL (r2 ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption.

Activation of ryanodine receptors is required for PKA-mediated downregulation of A-type K+ channels in rat hippocampal neurons.

A-type K+ channels (IA channels) contribute to learning and memory mechanisms by regulating neuronal excitabilities in the CNS, and their expression level is targeted by Ca2+ influx via synaptic NMDA receptors (NMDARs) during long-term potentiation (LTP). However, it is not clear how local synaptic Ca2+ changes induce IA downregulation throughout the neuron, extending from the active synapse to the soma. In this study, we tested if two major receptors of endoplasmic reticulum (ER), ryanodine (RyRs), and IP3 (IP3 R) receptors, are involved in Ca2+ -mediated IA downregulation in cultured hippocampal neurons of rats. The downregulation of IA channels was induced by doubling the Ca2+ concentration in culture media (3.6 mM for 24 hrs) or treating with glycine (200 μM for 3 min) to induce chemical LTP (cLTP), and the changes in IA peaks were measured electrophysiologically by a whole-cell patch. We confirmed that Ca2+ or glycine treatment significantly reduced IA peaks and that their effects were abolished by blocking NMDARs or voltage-dependent Ca2+ channels (VDCCs). In this cellular processing, blocking RyRs (by ryanodine, 10 μM) but not IP3 Rs (by 2APB, 100 μM) completely abolished IA downregulation, and the LTP observed in hippocampal slices was more diminished by ryanodine rather than 2APB. Furthermore, blocking RyRs also reduced Ca2+ -mediated PKA activation, indicating that sequential signaling cascades, including the ER and PKA, are involved in regulating IA downregulation. These results strongly suggest a possibility that RyR contribution and mediated IA downregulation are required to regulate membrane excitability as well as synaptic plasticity in CA3-CA1 connections of the hippocampus. © 2017 Wiley Periodicals, Inc.

Neural depolarization triggers Mg2+ influx in rat hippocampal neurons

Homeostasis of magnesium ion (Mg2+) plays key roles in healthy neuronal functions, and deficiency of Mg2+ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg2+ concentration ([Mg2+]i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg2+]i. The aim of this study is to explore the direct relationship between neural activity and [Mg2+]i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg2+]i and [Ca2+]i dynamics were simultaneously visualized with a highly selective fluorescent Mg2+ probe, KMG-104, and a fluorescent Ca2+ probe, Fura Red, respectively. [Mg2+]i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg2+]i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K+ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca2+ increase. Furthermore, the [Mg2+]i increase was abolished in Mg2+-free extracellular medium, indicating [Mg2+]i increase is due to Mg2+ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na+ channel opener, induced [Mg2+]i increase, and this [Mg2+]i increase was suppressed by the pretreatment of a non-specific Mg2+ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg2+]i increase results from Mg2+ influx through 2-APB-sensitive channels in rat hippocampal neurons.

Hypoxia Induces an Increase in Intracellular Magnesium via Transient Receptor Potential Melastatin 7 (TRPM7) Channels in Rat Hippocampal Neurons in Vitro

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.

Voltage-gated calcium channels are not affected by the novel anti-epileptic drug lacosamide

The novel anti-epileptic drug lacosamide targets two proteins - voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP-2) - suggesting dual modes of action for lacosamide. We recently identified the neurite outgrowth and axonal guidance protein CRMP-2 as a novel partner and regulator of the presynaptic N-type voltage-gated Ca(2+) channel (CaV2.2) [Brittain et al., J. Biol. Chem. 284: 31375-31390 (2009)]. Here we examined the effects of lacosamide on voltage-gated Ba(2+) channels. Lacosamide did not affect Ba(2+) currents via N- and P/Q- channels in rat hippocampal neurons or L-type Ca(2+) channels in a mouse CNS neuronal cell line, respectively. N-type Ba(2+) currents, augmented by CRMP-2 expression, were also unaffected by acute or chronic lacosamide exposure. These results establish that the anti-epileptic mode of action of lacosamide does not involve these voltage-gated Ca(2+) channels.

Involvement of protein kinase C and protein kinase A in the enhancement of L-type calcium current by GABA B receptor activation in neonatal hippocampus

In the early neonatal period activation of GABA B receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of G i/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6–8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of G i/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABA B agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the G i/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF- 109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19–36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6–22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM–1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABA B receptor mediated effect.

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca(2+)](i)) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca(2+)](i) from 118 +/- 21 to 440 +/- 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL's epileptogenic process.

Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca2+ channels in rat hippocampal neurons

Vascular endothelial growth factor (VEGF) protects neurons against ischemic injury. An overload of intracellular calcium ions (Ca(2+)) caused by the excessive release of glutamate is widely considered to be one of the molecular mechanisms of ischemic neuronal death. In the present study, we investigated whether VEGF could modulate the activity of Ca(2+) channels on the neuronal membrane. We used the Fluo-3 image method assisted by confocal laser scan microscopy to detect any Ca(2+) influx in primary cultured hippocampal neurons. Whole-cell patch-clamp techniques were used to record the activity of the high-voltage-activated (HVA) Ca(2+) currents in the CA1 pyramidal neurons of hippocampal slices that were freshly prepared from neonatal brains of rats. The results obtained from the Fluo-3 image experiments showed that VEGF pretreatment of cultured neurons at a final concentration of 50, 100, or 200 ng/ml acutely and dose dependently attenuated the Ca(2+) influx induced by application of KCl (60 mM) or glutamate (50 microM). This effect was blocked by SU1498, an antagonist of Flk-1 VEGF receptor. The influx of Ca(2+) returned to basal levels after removal of VEGF. Furthermore, electrophysiological recording data showed that VEGF could acutely reduce the amplitudes of the HVA Ca(2+) currents in a dose- and voltage-dependent manner. The HVA Ca(2+) currents also returned to the levels of the control after removal of VEGF from the system. Taken together, the results obtained from the present study demonstrated that VEGF specifically reduced the influx of Ca(2+) via the inhibitory activity of the HVA Ca(2+) channels in hippocampal neurons.

Discovery of talatisamine as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons

Blocking specific K+ channels has been proposed as a promising strategy for the treatment of neurodegenerative diseases. Using a computational virtual screening approach and electrophysiological testing, we found four Aconitum alkaloids are potent blockers of the delayed rectifier K+ channel in rat hippocampal neurons. In the present study, we first tested the action of the four alkaloids on the voltage-gated K+, Na+ and Ca2+ currents in rat hippocampal neurons, and then identified that talatisamine is a specific blocker for the delayed rectifier K+ channel. External application of talatisamine reversibly inhibited the delayed rectifier K+ current (IK) with an IC50 value of 146.0±5.8 μM in a voltage-dependent manner, but exhibited very slight blocking effect on the voltage-gated Na+ and Ca2+ currents even at the high concentration of 1–3 mM. Moreover, talatisamine exerted a significant hyperpolarizing shift of the steady-state activation, but did not influence the steady state inactivation of IK and its recovery from inactivation, suggesting that talatisamine had no allosteric action on IK channel and was a pure blocker binding to the external pore entry of the channel. Our present study made the first discovery of potent and specific IK channel blocker from Aconitum alkaloids. It has been argued that suppressing K+ efflux by blocking IK channel may be favorable for Alzheimer's disease therapy. Talatisamine can therefore be considered as a leading compound worthy of further investigations.

Binding of novel rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having in vitro neuroprotective activity. WYE-592 and ILS-920, synthesized by modifying rapamycin at the mTOR binding region, showed reduced immunosuppressive activity and enhanced neuroprotective activities. Also, ILS-920 demonstrated efficacy in an in vivo stroke model. This stimulated our interest in identifying potential targets that may be important in post-ischemic recovery. Affinity precipitation from a dorsal root ganglia/neuroblastoma (F11) cell lysate revealed that ILS-920 predominantly binds to FKBP52 and the β-subunit of L-type voltage dependent Ca2+channels. Further, kinetic analysis demonstrated that ILS-920 had a 972-fold higher selectivity for FKBP52 vs. FKBP12 than that of rapamycin. The involvement of FKBP52 and the β-subunit in the mechanism of neurite outgrowth was established by gene deletions. Electrophysiological analysis showed that IL-920 reduced 49% of total Ca2+ current, and inhibited the L-type Ca2+channels in rat hippocampal neurons, in contrast to rapamycin. These data suggest that this dual functionality- FKBP52 binding that stimulates neurite outgrowth perhaps via modulation of glucocorticoid and other steroid receptors, and inhibition of Ca2+ channel function to prevent neuronal cell death- may contribute to the compound's efficacy in stroke models.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca2+-activated K+ channel

Large conductance voltage and calcium-activated K+ channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane β2 subunit, KCNMB2, with the human pore-forming α subunit of the large conductance voltage and Ca2+-activated K+ channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca2+-dependent inactivation of large conductance Ca2+-activated K+ (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721–734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane β-subunit homolog. Proc Natl Acad Sci U S A 96:4137–4142]. Herein, we report that coexpression of β2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, β2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. β2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo’s intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping β2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. β2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of β2 that can control expression levels of hSlo. In contrast, a β2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, β2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of β2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

Light-activated ion channels for remote control of neuronal firing

Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K(+) channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K(+) (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.

Effect of β-estradiol on voltage-gated Ca 2+ channels in rat hippocampal neurons: a comparison with dehydroepiandrosterone

We investigated the effects of β-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate on intracellular calcium concentration ([Ca 2+] i) increases induced by γ-aminobutyric acid (GABA), high K + and N-methyl- d-aspartate acid (NMDA) in cultured hippocampal neurons. Acute treatment with β-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate inhibited the GABA-induced [Ca 2+] i increases to the similar extent. Tamoxifen, an estrogen receptor antagonist, did not block the inhibitory effects of β-estradiol. On the other hand, GABA type A (GABA A) receptor antagonists, picrotoxin and bicuculline, blocked the GABA-induced [Ca 2+] i increases. Previously, we demonstrated that GABA- and high K +-induced [Ca 2+] i increases were commonly mediated by voltage-gated calcium channels (VGCCs). Therefore, we examined the effects of these steroids on the high K +-induced [Ca 2+] i increases. The inhibitory effect of β-estradiol on the high K +-induced [Ca 2+] i increases was much greater than that of dehydroepiandrosterone and dehydroepiandrosterone sulfate. β-Estradiol inhibited the NMDA-induced [Ca 2+] i increases with an IC 50 of 51.8 μM and NMDA responses were reduced to half in the presence of 10 μM nifedipine, indicating that the NMDA-induced [Ca 2+] i increases also involved VGCCs. Further, we examined the inhibitory effect of β-estradiol on the high K +-induced [Ca 2+] i increases in the presence of a N-type VGCCs antagonist, 1 μM ω-conotoxin, or a L-type VGCCs antagonist, 10 μM nifedipine. The IC 50 value of β-estradiol alone (45.5 μM) was similar to that of ω-conotoxin (33.1 μM), while the value combined with nifedipine was reduced to 2.2 μM. β-Estradiol also abolished the positive modulatory effect of L-type VGCCs agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (Bay K 8644). Our results showed that the inhibitory mechanism of β-estradiol is different from that of dehydroepiandrosterone and dehydroepiandrosterone sulfate and β-estradiol may act primarily at L-type VGCCs.

Temperature dependence of pyrethroid modification of single sodium channels in rat hippocampal neurons.

Pyrethroid modulation of sodium channels is unique in the sense that it is highly dependent on temperature, the potency being augmented by lowering the temperature. To elucidate the mechanisms underlying the negative temperature dependence of pyrethroid action, single sodium channel currents were recorded from cultured rat hippocampal neurons using the inside-out configuration of patch-clamp technique, and the effects of the pyrethroid tetramethrin were compared at 22 and 12 degrees C. Tetramethrin-modified sodium channels opened with short closures and/or transitions to subconductance levels at 22 and 12 degrees C. The time constants of the burst length histograms for tetramethrin-modified channels upon depolarization to -60 mV were 7. 69 and 14.46 msec at 22 and 12 degrees C, respectively (Q(10) = 0. 53). Tetramethrin at 10 microm modified 17 and 23% of channels at 22 and 12 degrees C, respectively, indicating that the sensitivity of the sodium channel of rat hippocampal neurons to tetramethrin was almost the same as that of tetrodotoxin-sensitive sodium channels of rat dorsal root ganglion neurons and rat cerebellar Purkinje neurons. The time constants for burst length in tetramethrin-modified sodium channels upon repolarization to -100 mV from -30 mV were 8.26 and 68. 80 msec at 22 and 12 degrees C (Q(10) = 0.12), respectively. The prolongation of tetramethrin-modified whole-cell sodium tail currents upon repolarization at lower temperature was ascribed to a prolongation of opening of each channel. Simple state models were introduced to interpret behaviors of tetramethrin-modified sodium channels. The Q(10) values for transition rate constants upon repolarization were extremely large, indicating that temperature had a profound effect on tetramethrin-modified sodium channels.

Different distribution of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat hippocampal neurons.

The distribution patterns of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat primary cultured hippocampal neurons were investigated with a confocal laser-scanning microscope. Cells were loaded with the calcium indicator dye Oregon Green/AM, and the responses to high potassium (90 mM) solution with and without the existence of L- and N-type Ca2+ channel blockers were imaged. In general, extracellular application of high [K+] solution induced a [Ca2+]o-dependent increase of [Ca2+]i in both somata and dendritic processes. The increase was reduced by a N-type channel blocker, omega-conotoxin GVIA, and the reduction was greater in dendritic processes than in somata and proximal dendrites. In contrast, the reduction induced by the L-type calcium channel blocker, nifedipine, was observed evenly all over the neurons. The results demonstrated the heterogeneous distribution of nifedipine- and omega-conotoxin GVIA-sensitive calcium channels in cultured hippocampal neurons.

Ca2+ store-dependent potentiation of Ca2+-activated non-selective cation channels in rat hippocampal neurones in vitro.

1. Potentiation of calcium-activated non-selective cation (CAN) channels was studied in rat hippocampal neurones. CAN channels were activated by IP3-dependent Ca2+ release following metabotropic glutamate receptor (mGluR) stimulation either by Schaffer collateral input to CA1 neurones in brain slices in which ionotropic glutamate and GABAA receptors, K+ channels, and the Na+-Ca2+ exchanger were blocked or by application of the mGluR antagonist ACPD in cultured hippocampal neurones. 2. The CAN channel-dependent depolarization (DeltaVCAN) was potentiated when [Ca2+]i was increased in neurones impaled with Ca2+-containing microelectrodes. 3. Fura-2 measurements revealed a biphasic increase in [Ca2+]i when 200 microM ACPD was bath applied to cultured hippocampal neurones. This increase was greatly attenuated in the presence of Cd2+. 4. Thapsigargin (1 microM) caused marked potentiation of DeltaVCAN in CA1 neurones in the slices and of the CAN current (ICAN) measured in whole cell-clamped cultured hippocampal neurones. 5. Ryanodine (20 microM) also led to a potentiation of DeltaVCAN while neurones pretreated with 100 microM dantrolene failed to show potentiation of DeltaVCAN when impaled with Ca2+-containing microelectrodes. 6. The mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide m-chlorophenyl hydrazone (2 microM) also caused a potentiation of DeltaVCAN. 7. CAN channels are subject to considerable potentiation following an increase in [Ca2+]i due to Ca2+ release from IP3-sensitive, Ca2+-sensitive, or mitochondrial Ca2+ stores. This ICAN potentiation may play a crucial role in the 'amplification' phase of excitotoxicity.

Rundown of somatodendritic N-methyl-D-aspartate (NMDA) receptor channels in rat hippocampal neurones: evidence for a role of the small GTPase RhoA.

Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels. The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases. In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA. Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP. Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect. We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin.