In vitro electrophysiology of rat subicular bursting neurons

Intracellular recordings were used to study the electrophysiological properties of rat subicular neurons in a brain slice preparation in vitro. Cells were classified as bursting neurons (n = 102) based on the firing pattern induced by depolarizing current pulses. The bursting response recorded at resting membrane potential (−66.1 ± 6.2 mV, mean ± SD n = 94) was made up of a cluster of fast action potentials riding on a slow depolarization and was followed by an afterhyperpolarization. Tonic firing occurred at a membrane potential of approximately −55 mV. A burst also occurred upon termination of a hyperpolarizing current pulse. Tetrodotoxin (TTX, 1 μM) blocked the burst and decreased or abolished the underlying slow depolarization. These effects were not induced by the concomitant application of the Ca²⁺ channel blockers Co²⁺ (2 mM) and Cd²⁺ (1 mM). Subicular bursting neurons displayed voltage- and time-dependent inward rectifications of the membrane during depolarizing and hyperpolarizing current pulses. The inward rectification in the depolarizing direction was abolished by TTX, while that in the hyperpolarizing direction was blocked by extracellular Cs⁺ (3 mM), but not modified by Ba²⁺ (0.5–1 mM), TTX, or Co²⁺ and Cd²⁺. Tetraethylammonium (10 mM)-sensitive, outward rectification became apparent in the presence of TTX. These results suggest that neurons in the rat subiculum can display voltage-dependent bursts of action potentials as well as membrane rectification in the depolarizing and hyperpolarizing directions. These results also indicate that activation of a voltage-gated Na⁺ conductance may be instrumental in the initiation of bursting activity. Hippocampus 7:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Activation of ATP receptor increases the cytosolic Ca concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca²⁺ concentration ([Ca]_i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca²⁺ and Na⁺, and was blocked by voltage-dependent Ca²⁺ channel blockers. The results suggest that the ATP-induced depolarization increases Ca²⁺ influx resulting in the increase in [Ca]_i.     

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

Comparison of the electrophysiology and morphology of layers III and II neurons of the rat medial entorhinal cortex in vitro

The basic membrane characteristics of neurons in layers II and III of the medial entorhinal cortex (MEA) were recorded using the intracellular current clamp technique in in vitro slices of the rat brain. Two types of cells were distinguished according to the presence of a time-dependent inward rectification (SAG current) with hyperpolarizing current pulses. The cells in which this inward rectification was not observed (No-SAG cells) had a larger input resistance, a more negative resting membrane potential and a more depolarized firing threshold. They more often displayed a strongly adapting firing pattern, and their action potentials had a slower decay rate and lacked a depolarizing afterpotential, compared with the SAG cells. SAG cells typically had a prominent rebound depolarization at the end of a hyperpolarizing current and membrane potential oscillations (7 Hz) upon subthreshold depolarizations. Cs⁺ blocked the time-dependent inward rectification. The rebound depolarization persisted, even in the presence of tetrodotoxin. Biocytin labelling showed that layer III consisted mainly of pyramidal-shaped cells. Most layer III cells were of the No-SAG type. All cells in layer II, stellate and pyramidal cells, were classified as SAG cells. We conclude that the cells in MEA layers II and III display different electroresponsiveness, but that this appears to be more related to the layer where they are located than to a specific morphology. As layer III consisted mainly of cells of the No-SAG type, we suggest that layer III cells are less excitable than the SAG type layer II cells.     

Relation of exocytosis and Ca-activated K current during Ca release from intracellular stores in individual rat chromaffin cells

Measurement of the change in cell membrane capacitance (C_m) along with the change in I_K(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca²⁺-free external solution, bradykinin (100 nM) caused a transient increase in C_m with a concurrent change in I_K(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP₃-mediated increase of submembrane-free Ca²⁺. The increases in C_m and I_K(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca²⁺-free solution, and this was irreversibly blocked by ryanodine (1 μM) in a use-dependent manner. Caffeine-sensitive intracellular Ca²⁺ stores were also depleted in several seconds and recovered by an influx of external Ca²⁺. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca²⁺ release from the same or distinct but rapidly equilibrating intracellular Ca²⁺ stores. The single cell assay of exocytosis and the increase in I_K(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca²⁺ release from intracellular stores potentially increases submembrane Ca²⁺ concentration and modulates simultaneously two submembrane Ca²⁺-dependent processes, exocytosis and I_K(Ca), in rat adrenal chromaffin cells.     

Synthetic ω-conotoxin: a potent calcium channel blocking neurotoxin

The Ca²⁺ channel blocking action of synthetic ω-conotoxin (ωCTX) was studied on isolated frog dorsal root ganglion neurons using a ‘concentration clamp’ technique which enabled internal perfusion and rapid external solution change. At 100 nM, ωCTX showed a time-dependent depression of Ca²⁺ current (I_Ca). At higher concentrations, ωCTX exhibited a dose-dependent depression of I_Ca amplitude without changing the current-voltage relationship. Increases in external Ca²⁺ concentration partly overcame the inhibitory action of ωCTX on the I_Ca amplitude. At 10 μM ωCTX totally blocked I_Ca without effect on the Na⁺ current. It was likely that ωCTX had high selectivity for the Ca²⁺ channel.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Nitric oxide as intracellular modulator: internal production of NO increases neuronal excitability via modulation of several ionic conductances

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO‐producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)‐expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single‐cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long‐term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I_NaP), voltage‐gated Ca currents (I_Ca) and small‐conductance calcium‐activated potassium (SK) channels. We show that NOS inhibitors 7‐nitroindazole and S‐methyl‐l ‐thiocitrulline resulted in a decrease in I_NaP, and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I_NaP, riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I_Ca, and blocked spontaneous and depolarization‐induced spiking, suggesting that intrinsic NO controlled I_Ca via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self‐produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.     

Ca-Antagonistic action of bevantolol on hypothalamic neurons in vitro: its comparison with those of other β-adrenoceptor antagonists, a local anesthetic and a Ca-antagonist

The Ca²⁺-antagonistic action of bevantolol, aβ₁-adrenoceptor antagonist, on high- and low-voltage activated Ca²⁺ currents (HVA- and LVA-I_Ca) was examined on neurons dissociated from rat brain. Bevantolol (10⁻⁶ to 10⁻⁴ M) inhibited concentration-dependently bothI_Ca. The IC₅₀ value of bevantolol for LVA-I_Ca was 4 × 10⁻⁵ M, while bevantolol at 10⁻⁴ M inhibited HVA-I_Ca by 28.5 ± 7.7%. The potency of bevantolol in inhibiting bothI_Ca was greater than those of propranolol, labetalol and lidocaine, while the inhibitory action of bevantolol on voltage-activated Na⁺ current was weakest among them. Bevantolol may possess Ca²⁺-antagonistic action that is independent from local anesthetic action.     

Metabotropic glutamate receptor agonist ACPD inhibits some,but not all,muscarinic-sensitive K+ conductances in basolateral amygdaloid neurons

Muscarinic agonists produce membrane depolarization and losses of spike frequency accommodation and the slow afterhyperpolarization (AHP) when applied to neurons of the basolateral amygdala (BLA). Underlying these changes are the muscarinic-induced inhibitions of several K⁺ conductances, including the voltage-activated M-current (I_M), a slowly decaying Ca²-activated current (I_AHP), a voltage-insensitive leak current (I_Leak), and the hyperpolarization-activated inward rectifier current (I_IR) Similar depolarizations and losses of the slow AHP have been observed in other neuronal cell types following stimulation of metabotropic glutamate receptors. Therefore, we tested the effects of the metabotropic glutamate receptor agonist, 1-aminocyclopentanels, 3r-dicarboxylic acid (ACPD), on pyramidal neurons impaled with a single microelectrode for current- and voltage-clamp recordings in a brain slice preparation of the rat BLA. Application of ACPD (20 or 100 μM) to BLA neurons inhibited I_M and I_{A HP}, resulting in membrane depolarization and reductions in the amplitude and duration of the slow AHP. However, ACPD did not inhibit the muscarinic-sensitive current I_IR, nor was I_Leak blocked in the majority of neurons examined. These findings suggest the possibility that muscarinic cholinergic and metabotropic glutamatergic receptor agonists may activate separate intracellular transduction pathways which have convergent inhibitory effects onto I_M and I_AHP in BLA pyramidal neurons. © 1994 Wiley-hiss, Inc.     

Rectification Properties and Ca2+ Permeability of Glutamate Receptor Channels in Hippocampal Cells

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na⁺ and K⁺, while Ca²⁺ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca²⁺ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca²⁺ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca²⁺ . Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca²⁺ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca²⁺-impermeable (P_caIP_cs= 0.1) while type II cells (6.5%) expressed Ca²⁺-permeable receptors (P_caIP_cs=0.9).Type III cells (41.3%) showed responses with low but not negligible Ca²⁺ permeability (P_caIP_cs= 0.18). The degree of Ca²⁺ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca²⁺ permeability. In acutely dissociated neurons, the restricted activation of the receptors located either in dendritic or somatic membranes revealed that inward rectifying (i.e. Ca²⁺-permeable) AMPA receptors are preferentially located in the dendritic shaft (i.e. synaptic field). Our results indicate that oligomeric AMPA receptors of different subunit composition are coexpressed in dissimilar proportions in different cells, which would explain the incomplete inward rectification and graded Ca²⁺ permeability. In addition, Ca²⁺-permeable AMPA receptors may exhibit non-homogeneous subcellular distribution.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

Bombesin facilitates GABAergic transmission and depresses epileptiform activity in the entorhinal cortex

Bombesin and the bombesin‐like peptides including neuromedin B (NMB) and gastrin‐releasing peptide (GRP) are important neuromodulators in the brain. We studied their effects on GABAergic transmission and epileptiform activity in the entorhinal cortex (EC). Bath application of bombesin concentration‐dependently increased both the frequency and amplitude of sIPSCs recorded from the principal neurons in the EC. Application of NMB and GRP exerted the same effects as bombesin. Bombesin had no effects on mIPSCs recorded in the presence of TTX but slightly depressed the evoked IPSCs. Omission of extracellular Ca²⁺ or inclusion of voltage‐gated Ca²⁺ channel blockers, Cd²⁺ and Ni²⁺, blocked bombesin‐induced increases in sIPSCs suggesting that bombesin increases GABA release via facilitating extracellular Ca²⁺ influx. Bombesin induced membrane depolarization and slightly increased the input resistance of GABAergic interneurons recorded from layer III of the EC. The action potential firing frequency of the interneurons was also increased by bombesin. Bombesin‐mediated depolarization of interneurons was unlikely to be mediated by the opening of a cationic conductance but due to the inhibition of inward rectifier K⁺ channels. Bath application of bombesin, NMB and GRP depressed the frequency of the epileptiform activity elicited by deprivation of Mg²⁺ from the extracellular solution suggesting that bombesin and the bombesin‐like peptides have antiepileptic effects in the brain. © 2013 Wiley Periodicals, Inc.     

Effect of KB-2796, a new diphenylpiperazine Ca antagonist, on voltage-dependent Ca currents and oxidative metabolism in dissociated mammalian CNS neurons

The effects of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine-2HCl, on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) and on oxidative metabolism were studied in neurons freshly dissociated from rat brain. KB-2796 reduced the peak amplitude of LVA I_Ca in a concentration-dependent manner with a threshold concentration of 10⁻⁷ M when the LVA I_Ca was elicited every 30 s in the external solution with 10 mM Ca²⁺. The concentration for half-maximum inhibition (IC₅₀) was 1.9 × 10⁻⁶M. At 10⁻⁵ M or more of KB-2796, a complete suppression of the LVA I_Ca was observed in the majority of neurons tested. There was no apparent effect on the current-voltage (I-V) relationship and the current kinetics. KB-2796 delayed the reactivation and enhanced the inactivation of the Ca²⁺ channel for LVA I_Ca voltage- and time-dependently, suggesting that KB-2796 preferentially binds to the inactivated Ca²⁺ channel. KB-2796 at a concentration of3.0 × 10⁻⁶M also decreased the peak amplitude of the HVA I_Ca without shifting the I-V relationship. In addition, KB-2796 reduced the oxidative metabolism (the formation of reactive oxygen species) of the neuron in a concentration-dependent manner with a threshold concentration of3 × 10⁻⁶M. It is suggested that the inhibitory action of KB-2796 on the neuronal Ca²⁺ influx and the oxidative metabolism, in combination with a cerebral vasodilatory action, may reduce ischemic brain damage.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

A Study of the Barium-sensitive and -insensitive Components of the Action of Thyrotropin-releasing Hormone on Lumbar Motoneurons of the Rat Isolated Spinal Cord

The electrophysiological action of thyrotropin-releasing hormone (TRH) on rat spinal motoneurons was studied in vitro using single-electrode voltage- and current-clamp techniques. In current-clamp conditions TRH elicited a slowly developing depolarization, associated with a large input resistance increase and sustained neuronal firing; the primary metabolites of TRH were ineffective. Under voltage-clamp conditions in the presence of tetrodotoxin, TRH evoked a large inward current (I_TRH; peaking at approximately –40 mV) associated with a large input conductance fall. Only 44% of cells displayed I_TRH reversal; when the chord conductance values of these cells were plotted against membrane potential, a bell-shaped relation occurred, indicating voltage-dependent block by TRH of a persistent conductance active over a wide range of membrane potentials. I_TRH reversal values were shifted to more positive levels in high K⁺ solution in Nernstian fashion; hence a large proportion of the TRH response is suggested to be mediated by the block of a K⁺ conductance (I_K(T)). I_K(T) (and its voltage-dependent block by TRH) was resistant to certain K⁺ channel antagonists (tetraethylammonium, Cs⁺, 4-aminopyridine or apamin), but was depressed by Ba²⁺. The Ba²⁺-resistant fraction of I_TRH was attenuated by Cd²⁺, Mn²⁺ or Co²⁺, indicating that it probably involved a Ca²⁺-sensitive inward current. Concomitant application of Ba²⁺ and Cd²⁺ induced a near-total block of the response to TRH. It is suggested that suppression of I_K(T), associated with the onset of a Ca²⁺-sensitive current, can explain the excitatory effect of TRH on rat spinal motoneurons.