Glutamine-induced membrane currents in cultured rat hippocampal neurons

Glutamine is present at high concentrations in the extracellular fluid of the brain. It shuttles between glia cells and neurons, and serves as a precursor for both glutamate and γ-amino butyric acid. Direct actions of glutamine at central neurons are, however, not well understood. Here we showed that l -glutamine (0.5–10 m m ) evoked a dose-dependent inward transmembrane current in primarily cultured rat hippocampal neurons. Typical responses were outwardly rectifying and had a reversal potential around 0 mV. The current was partially sensitive towards blockers of ionotropic glutamate receptors and was partially carried by activation of N -methyl- d -aspartate receptors. However, cellular responses to l -glutamine showed clear biophysical and pharmacological differences to l -glutamate-evoked currents. Responses were highly specific for l -glutamine and no responses could be evoked by d -glutamine, l -alanine, l -valine, l -leucine and the system-A-specific agonist α-(methylamino)-isobutyric acid. Together, these data indicate that hippocampal neurons can be depolarized by electrogenic effects specific for l -glutamine.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

We studied the effects of hypertonia on voltage-gated currents of freshly isolated hippocampal CA1 neurons, using open pipette whole-cell as well as gramicidin-perforated patch-clamp recording. Extracellular osmolarity (π_o) was raised by adding mannitol (50 or 100 mmol/l) to the bathing solution. Hypertonia depressed voltage-gated sodium, potassium and calcium currents in all trials. The threshold activation voltage of the currents did not change during hypertonic depression, but maximal activation of Ca²⁺ current shifted to a more negative potential, suggesting stronger depression of high- compared to low-voltage activated currents. During 30 min high π_o treatment (recorded with open pipette), the depression reached maximum in 10–15 min of exposure. The depression of the computed transient component of the K⁺ current recorded by open pipette was statistically not significant. Following hypertonic treatment recovery of the I_Na, the sustained I_K and sustained I_Ca were incomplete compared to control cells maintained in normal solution for an equal length of time. In hippocampal tissue slices hypertonia (+25, +50 and +100 mmol/l fructose) reversibly depressed excitatory postsynaptic currents (EPSCs). We conclude that the shutdown of membrane ion currents by elevated π_o is not selective, but the degree of the suppression varies among current types. Raising π_o in human patients, possibly combined with mild artificial acidosis, may be useful in the prevention and treatment of acute crises associated with excessive excitation or depolarization of neurons.     

Miniature excitatory synaptic currents in cultured hippocampal neurons.

We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons. In bathing solutions containing tetrodotoxin (TTX), the cells showed spontaneous inward currents (SICs) ranging in size from 1 to 100 pA. Several observations indicated that the SICs were miniature excitatory synaptic currents mediated primarily by non-NMDA (N-methyl-D-aspartate) excitatory amino acid receptors: the rising phase of SICs was fast (1 ms to half amplitude at room temperature) and smooth, suggesting unitary events. The SICs were blocked by the broad-spectrum glutamate receptor antagonist gamma-D-glutamylglycine (DGG), but not by the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (5-APV). SICs were also blocked by desensitizing concentrations of quisqualate. Incubating cells in tetanus toxin, which blocks exocytotic transmitter release, eliminated SICs. The presence of SICs was consistent with the morphological arrangement of glutamatergic innervation in the cell cultures demonstrated immunohistochemically. Spontaneous outward currents (SOCs) were blocked by bicuculline and presumed to be mediated by GABAA receptors. This is consistent with immunohistochemical demonstration of GABAergic synapses. SIC frequency was increased in a calcium dependent manner by bathing the cells in a solution high in K+, and application of the dihydropyridine L-type calcium channel agonist BAY K 8644 increased the frequency of SICs. Increases in SIC frequency produced by high K+ solutions were reversed by Cd2+ and omega-conotoxin GVIA, but not by the selective L-type channel antagonist nimodipine. This suggested that presynaptic L-type channels were in a gating mode that was not blocked by nimodipine, and/or that another class of calcium channel makes a dominant contribution to excitatory transmitter release.     

Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons

The present study examined the effects of glutamate on the outgrowth of dendrites and axons in isolated hippocampal pyramidal-like neurons in cell culture. During the first day of culture the survival and outgrowth of these neurons was unaffected by high concentrations (up to 1 nM) of glutamate, quisqualic acid (QA), kainic acid (KA), and N-methyl-D-aspartic acid. Beginning on day 2 of culture high levels of glutamate, KA and QA were toxic to the majority of pyramidal neurons, while subtoxic levels of these agents caused a well-defined, dose-dependent, sequence of effects on dendritic outgrowth. At increasing concentrations of glutamate, QA, and KA, the following events were observed: (1) dendritic outgrowth rates were reduced, while axonal elongation rates were unaffected; (2) dendritic length was reduced, while axons continued to grow; (3) dendrites regressed dramatically, and axonal outgrowth rate was reduced. These dendrite-specific effects of glutamate were apparently mediated at the growth cones since focal application of glutamate to individual dendritic growth cones resulted in suppression of growth cone activity and a regression of the dendrite; axons were unaffected by focal glutamate application. Pharmacological tests using glutamate receptor agonists and antagonists demonstrated that receptors of the KA/QA type mediated the glutamate effects on outgrowth and survival. The calcium channel blocker Co2+ prevented both glutamate neurotoxicity and glutamate-induced dendritic regression. Ionophore A23187 and elevations in extracellular K+ levels each caused a dose-dependent series of outgrowth and survival responses similar to those caused by glutamate. Taken together, these results indicate that activation of glutamate receptors leads to the opening of voltage-dependent calcium channels; the resulting increases in calcium influx lead to the observed alterations in dendritic outgrowth and neuronal survival.     

Delayed rectifier currents in rat globus pallidus neurons are attributable to Kv2.1 and Kv3.1/3.2 K(+) channels.

The symptoms of Parkinson disease are thought to result in part from increased burst activity in globus pallidus neurons. To gain a better understanding of the factors governing this activity, we studied delayed rectifier K(+) conductances in acutely isolated rat globus pallidus (GP) neurons, using whole-cell voltage-clamp and single-cell RT-PCR techniques. From a holding potential of -40 mV, depolarizing voltage steps in identified GP neurons evoked slowly inactivating K(+) currents. Analysis of the tail currents revealed rapidly and slowly deactivating currents of similar amplitude. The fast component of the current deactivated with a time constant of 11. 1 +/- 0.8 msec at -40 mV and was blocked by micromolar concentrations of 4-AP and TEA (K(D) approximately 140 microM). The slow component of the current deactivated with a time constant of 89 +/- 10 microseconds at -40 mV and was less sensitive to TEA (K(D) = 0.8 mM) and 4-AP (K(D) approximately 6 mM). Organic antagonists of Kv1 family channels had little or no effect on somatic currents. These properties are consistent with the hypothesis that the rapidly deactivating current is attributable to Kv3.1/3.2 channels and the slowly deactivating current to Kv2.1-containing channels. Semiquantitative single-cell RT-PCR analysis of Kv3 and Kv2 family mRNAs supported this conclusion. An alteration in the balance of these two channel types could underlie the emergence of burst firing after dopamine-depleting lesions.     

Rat supraoptic neurons are resistant to glutamate neurotoxicity.

Magnocellular neurosecretory cells of the supraoptic nucleus (SON) are thought to be endogenously resistant to glutamate toxicity. In this study, we sought physiological and morphological evidence of this resistance in rats that received multiple peri-nuclear injections of ibotenate. In this preparation, ibotenate produced a large necrotic zone encompassing the SON but not within the nucleus itself. Extracellular recordings in vivo from 68 'spared' SON neurons from lesioned rats revealed normal patterns of electrical activity. Intracellular analysis in vitro from 13 'spared' SON neurons indicated that their intrinsic membrane properties, osmosensitivity and spontaneous synaptic activity did not differ significantly from that of controls. We conclude that SON neurons retain both a morphological and a physiological resistance to glutamate neurotoxicity.     

Miniature release events of glutamate from hippocampal neurons are influenced by the dystonia-associated protein torsinA

TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in‐frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal‐dominant, early‐onset generalized DYT1 dystonia. Although only 30–40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE‐torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild‐type, heterozygous, and homozygous ΔE‐torsinA knock‐in mice using two approaches. In the first approach, patch‐clamp electrophysiology was used to record glutamate‐mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA_A receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild‐type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild‐type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild‐type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE‐torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect. Synapse 66:807–822, 2012. © 2012 Wiley Periodicals, Inc.     

Turtle cortical neurons survive glutamate exposures that are lethal to mammalian neurons

Glutamate is an excitatory neurotransmitter in turtle and mammalian cortex. In high concentrations it is toxic to mammalian neurons and is an important mediator in the pathway that leads to neuronal death from anoxia. Turtle neurons are remarkably resistant to anoxic injury and we sought to determine whether part of this resistance could be attributed to the sensitivity of turtle neurons to glutamate toxicity. Embryonic turtle cortical neurons were grown for 25 days in dissociated cell culture using a modification of a method developed for murine cortical cell culture. Turtle neurons in dissociated culture were found to express glutamate receptors which include bothN-methyl-d-aspartate (NMDA) and non-NMDA receptor types. Remarkably, these neurons survive 5 minute exposures to glutamate in concentrations up to 3 mM, doses 30 times the LD₅₀ and 6 times the LD₁₀₀ for mouse cortical neurons¹². Elucidating the mechanism for this resistance may suggest new strategies for brain protection.     

Reduced potassium currents in old rat CA1 hippocampal neurons

Potassium currents are an important factor in repolarizing the membrane potential and determining the level of neuronal excitability. We compared potassium currents in CA1 hippocampal neurons dissociated from young (2-3 months old) and old (26-30 months old) Sprague-Dawley rats. Whole-cell patch-clamp techniques were used to measure the delayed rectifier (sustained) and the A-type (transient) potassium currents. The delayed rectifier current was smaller in old (548 +/- 57 pA) than in young (1193 +/- 171 pA) neurons. In the absence of extracellular calcium, the delayed rectifier current was also smaller in old (427 +/- 41 pA) than in young (946 +/- 144 pA) neurons. The cell membrane capacitance was unchanged in old (13.3 +/- 1.2 pF) compared to young (13.6 +/- 1.2 pF). Therefore, the reduction in the delayed rectifier current was not due to a change in membrane surface area. Moreover, activation and inactivation of the delayed rectifier current were unchanged in old compared to young neurons. The slope of the current-voltage relation, however, was smaller in old (B = 5.03) than in young (B = 9.62) neurons. Similarly, the A-current was smaller in old (100 +/- 16 pA) than in young (210 +/- 44 pA) neurons in the presence of extracellular calcium. This reduction of potassium currents could account for the prolongation of action potentials reported previously for old rat CA1 hippocampal neurons. The age-related reduction in potassium current indicates plasticity in neuronal function that can impact communication in the hippocampal neural network during aging.     

Psychosine blocks quisqualate-induced glutamate excitotoxicity in hippocampal CA sector neurons

Quisqualate is a potent specific agonist for Group 1 metabotropic glutamate receptors (mGluR's), that activate G protein-coupled phospholipase C (PLC) in a molecular signal-transduction mechanism that raises cytoplasmic Ca²⁺ and, when excessive, damages hippocampal neurons. Psychosine (β-galactosylsphingosine), a cationic lysosphingolipid occurring naturally in nervous tissues, dose-dependently inhibited PLC activation induced by metabotropic α₁-adrenergic receptor signaling in cultured rat brain astrocytes in vitro. In the present study, we have tested neuroprotective efficacy of psychosine in vivo, in a rat model of glutamate excitotoxicity induced by intracerebroventricular (i.c.v.) administration of quisqualate. A sublethal i.c.v. dose of quisqualate caused episodes of prolonged akinesia and convulsions, and major damage to pyramidal neurons of the hippocampal CA1 and CA3 sector, but not to granule cell neurons of the dentate gyrus. Prior infusion of psychosine greatly attenuated quisqualate-induced behaviors, and fully prevented destruction by quisqualate of vulnerable hippocampal neurons. Psychosine may prove useful in prophylaxis of neurodegenerative disorders that arise from intensive hippocampal Group 1 mGluR stimulation.     

Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action of the Nephila clavata spider neurotoxin was studied using the concentration clamp method in isolated neurons of the rat hippocampus. Crude venom JSTX blocked L-glutamate-, quisqualate- and kainate-activated ionic currents mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Ionic currents elicited by all agonists were depressed by crude JSTX venom to 34-35% of its initial amplitude with no recovery during prolonged washing. An active fraction of JSTX venom blocked ionic currents almost completely, but its action was partially reversible. The concentration dependences of blocking kinetics allowed determining the rate constants of JSTX interaction with glutamate receptors. It is supposed that JSTX blocks the non-NMDA ionic channels in some of their open states and may be one of useful tools in further biochemical and electrophysiological characterization of the glutamate-mediated synaptic transmission.     

Phenformin suppresses calcium responses to glutamate and protects hippocampal neurons against excitotoxicity

Phenformin is a biguanide compound that can modulate glucose metabolism and promote weight loss and is therefore used to treat patients with type-2 diabetes. While phenformin may indirectly affect neurons by changing peripheral energy metabolism, the possibility that it directly affects neurons has not been examined. We now report that phenformin suppresses responses of hippocampal neurons to glutamate and decreases their vulnerability to excitotoxicity. Pretreatment of embryonic rat hippocampal cell cultures with phenformin protected neurons against glutamate-induced death, which was correlated with reduced calcium responses to glutamate. Immunoblot analyses showed that levels of the N-methyl-d-aspartate (NMDA) subunits NR1 and NR2A were significantly decreased in neurons exposed to phenformin, whereas levels of the AMPA receptor subunit GluR1 were unchanged. Whole-cell patch clamp analyses revealed that NMDA-induced currents were decreased, and AMPA-induced currents were unchanged in neurons pretreated with phenformin. Our data demonstrate that phenformin can protect neurons against excitotoxicity by differentially modulating levels of NMDA receptor subunits in a manner that decreases glutamate-induced calcium influx. These findings show that phenformin can modulate neuronal responses to glutamate, and suggest possible use of phenformin and related compounds in the prevention and/or treatment of neurodegenerative conditions.     

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.     

Glutamate toxicity in immature cortical neurons precedes development of glutamate receptor currents

Cationic fluxes resulting from glutamate receptor activity have recently been implicated in neurotoxicity. Immature cortical neurons are insensitive to the toxic effects of glutamate receptor stimulation. However, these neurons are killed by glutamate via a non-receptor-mediated mechanism thought to stem from glutamate's ability to inhibit cystine uptake. To examine the basis for their resistance to receptor-mediated toxicity, we have studied the development of glutamate receptor-mediated inward currents in cortical neurons in culture using the whole-cell voltage-clamp technique. We report that in immature cortical neurons (prepared from day-17 fetal brain and cultured for 1-3 days), N-methyl-D-aspartate, quisqualate, and glutamate are able to evoke only very small inward currents in a low percentage of neurons. After 7 days of culture, greater than 80% of neurons examined exhibited currents activated by these glutamate receptor agonists. Although most neurons expressed glutamate agonist-evoked currents after 7 days in culture, the amplitude of these currents was less than 10% of that observed after 15 days in culture. In contrast to currents activated by glutamate receptor agonists, those activated by gamma-aminobutyric acid reached maximal levels after only 2 days of culture. These results indicate that the delayed development of glutamate receptor-mediated currents accounts for the resistance of immature cortical neurons to glutamate receptor-mediated toxicity.     

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

A steroid anesthetic prolongs inhibitory postsynaptic currents in cultured rat hippocampal neurons

Whole-cell patch-clamp recordings were made from cultured rat hippocampal neurons to examine the effects of the steroidal general anesthetic alphaxalone (3 alpha-hydroxy 5 alpha-pregnane 11,20-dione) on responses to pharmacologically applied and physiologically released GABA. At low micromolar concentrations in the anesthetic range, alphaxalone potentiated Cl- conductance responses elicited by GABA and also prolonged evoked GABA-mediated postsynaptic potentials. Under voltage clamp at -40 mV, rapid outwardly directed synaptic currents were evoked that decayed with single exponential kinetics; mean decay time constant was 24 msec at room temperature. Alphaxalone prolonged the decay of these inhibitory postsynaptic currents by 5- to 8-fold, with no increase in peak amplitude or change in growth time. This substantial prolongation of GABA-mediated inhibitory synaptic conductance at clinically effective concentrations may contribute significantly to the anesthetic activity of alphaxalone.     

PACAP对谷氨酸引起海马神经元损伤的保护作用

目的研究垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对谷氨酸引起的海马神经元损伤的保护作用及其受体机制。方法海马神经元体外培养７ｄ,给予谷氨酸。结果当谷氨酸是０．１～１．０ｍｍｏｌ／Ｌ时,随着剂量的增加,神经元的存活率逐渐降低;１０－９ｍｏｌ／Ｌ～１０－１３ｍｏｌ／Ｌ的ＰＡＣＡＰ,能减轻谷氨酸引起的海马神经元损伤;ＰＡＣＡＰⅠ型受体特异性拮抗剂ＰＡＣＡＰ６－３８能抑制ＰＡＣＡＰ减轻谷氨酸对海马神经元损伤作用。结论ＰＡＣＡＰ具有减轻谷氨酸引起的海马神经元损伤的作用,该作用是由ＰＡＣＡＰⅠ型受体介导的。     

Conductance properties of GABA-activated chloride currents recorded from cultured hippocampal neurons

The conductance characteristics of gamma-aminobutyric acid-activated single channel currents from cultured hippocampal neurons were examined using patch clamp techniques. GABA-activated currents had amplitudes which were linearly correlated to the membrane potentials over a range of -80 to +70 mV and an open time and burst time of 2.2 and 4.3 ms, respectively. The conductance of the gamma-aminobutyric acid-activated channels was 19 pS. These data demonstrate that cultured hippocampal neurons have channel conductances which have characteristics different from those of adult neurons.