Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

Role of kainate receptor activation and desensitization on the [Ca(2+)](i) changes in cultured rat hippocampal neurons

We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca(2+)](i) caused by kainate showed cell-to-cell variability. The [Ca(2+)](i) increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca(2+)](i) changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca(2+)](i) and toxicity.     

Electrophysiological responses to muscarinic receptor stimulation in cultured hippocampal neurons.

Electrophysiological recordings of responses to muscarinic receptor stimulation in cultured embryonic hippocampal neurons have been largely unsuccessful to date. In this study muscarinic receptor binding was demonstrated in 2-week-old embryonic rat hippocampal cultures. Using whole-cell patch-clamp recording we found that 1-5 microM carbachol produced multiple effects including depolarization, increased action potential firing rate, increased synaptic activity and a reduction in the amplitude of medium-duration afterhyperpolarizations. Voltage-clamp analysis revealed a time-dependent current relaxation with hyperpolarizing steps from a holding potential of about -40 mV which was inhibited by 10 microM muscarine or 50 microM carbachol and had characteristics similar to those of the m-current. Both atropine and pirenzepine inhibited all of these effects indicating that these cholinergic actions were mediated by muscarinic receptors. This study shows that muscarinic responses obtained classically in hippocampal brain slices can also be produced in cultured hippocampus.     

Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons.

Opioid agonists hyperpolarize neurons of the locus coeruleus (LC) in the slice preparation. When opioids were applied at concentrations that caused a maximum hyperpolarization, the membrane potential hyperpolarized to a peak (about 30 mV) in the first minute and then declined over a period of 5 min. In addition, following the washout, the amplitude of the hyperpolarization induced by a lower concentration of opioid was significantly reduced as compared to control. The original response to both the low and the high concentrations of opioid recovered after removal of opioids for about 20 min. The decline in response, termed "acute desensitization," was observed only with concentrations of opioids that caused a maximum hyperpolarization and was dependent on the concentration of opioid applied (EC50 for [Met5]-enkephalin (ME), between 3 and 5 microM). The response to ME (300 nM) was reduced to 6% of control following washout of a 5-min application of ME (30 microM), whereas the response to noradrenaline (300 nM) was reduced to 75% of control. The acute desensitization therefore was selective for the opioid receptor with marginal cross-desensitization to the alpha 2-adrenoceptor-mediated hyperpolarization. The desensitization still occurred following treatment with beta-chlornaltrexamine (beta-CNA), to decrease receptor reserve, as well as in cells taken from animals treated chronically with morphine. The mechanism for the acute desensitization was investigated using agents thought to alter kinase activity. This acute desensitization may represent an initial stage in the development of tolerance produced by chronic administration of opioids.     

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.     

Complex responses activated by ibotenate in postnatal rat hippocampal neurons

The actions of the excitatory amino acid ibotenate were investigated in postnatal rat hippocampal neurons. In neurons voltage clamped at negative membrane potentials using low-chloride internal solutions, ibotenate responses consist of an inward cationic current and two outward currents. The inward current is inhibited by 2-amino-5-phosphonovalerate (APV), a selectiveN-methyl-d-aspartate (NMDA) antagonist. The two outward currents consist of a picrotoxin and bicuculline-sensitive chloride current and a slowly developing calcium activated potassium current. The bicuculline sensitive current appears to be the product of contamination of ibotenate samples with the γ-aminobutyric acid (GABA) agonist muscimol and not the result of a direct action of ibotenate.     

Maturation of vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons

Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 24 h increased from 3.6% in 3-day-old rats to >90% in rats older than 21 days. Time-lapse imaging of neuronal morphology following NMDA demonstrated increasingly prevalent and severe injury as a function of postnatal age. Neither high- nor low-affinity calcium dyes demonstrated differences in peak NMDA-induced [Ca(2+)](i) increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of [Ca(2+)](i) homeostasis. Because of the role of mitochondrial Ca(2+) buffering in [Ca(2+)](i) homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DeltaPsi) as a function of postnatal age. NMDA markedly dissipated DeltaPsi in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons, (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondrion.     

Quantal and subquantal GABAergic transmissions in cultured rat hippocampal neurons

At the neuromuscular junction, spontaneous miniature excitatory synaptic currents mediated by acetylcholine are considered elementary, “quantal” transmissions. These miniature conductances can be quantitatively dichotomized into a large-mode class whose mode is the mean of a normal, bell-shaped distribution and a small-mode class whose distribution is skewed to lower values with its mode being a fraction of the large-mode class. The large-mode class constitutes the population of synaptic signals originally utilized to formulate tenets of “quantal” transmission, which have been tacitly adopted in more recent studies of fast transmission at central synapses. Large- and small-mode conductance classes of inhibitory synaptic elementary conductances mediated by GABA have now been recorded in cultured hippocampal neurons (Vautrin J, Schaffner AE, Barker JL, 1991, Neurosci Lett 138:67). Pairs of hippocampal neurons were patch recorded at optimal signal-to-noise and, using time course analysis, two elementary fluctuations (0.1–0.3 nS and 1–2 nS) were found within synaptic conductances evoked either by presynaptic action potentials or by presynaptic terminal stimulation. These results were interpreted with a simple model that shows how different frequencies of unitary GABA release can generate either small-mode, skew-distributed conductance (0.5–3 kHz) or large-mode, normally-distributed conductances (≥ 10 kHz). Only the latter satisfies the original tenets of the classic quantal theory.     

Type-II cadherins modulate neural activity in cultured rat hippocampal neurons

Cadherins, cell adhesion molecules widely expressed in the nervous system, are thought to be involved in synapse formation and function. To explore the role of cadherins in neuronal activity, we performed electrophysiological and morphological analyses of rat hippocampal cultured neurons overexpressing type-II cadherins, such as cadherin-6B and cadherin-7. We found that cadherin-6B increased but cadherin-7 decreased the number of protrusions of dendritic spines, and affected the frequency of miniature excitatory postsynaptic currents. Our results suggest that type-II cadherins may modulate neural activity by regulating neuronal morphology.     

Electrophysiological properties of identified postnatal rat hippocampal pyramidal neurons in primary culture

Electrophysiological experiments were performed on primary cell cultures of retrogradely labelled postnatal rat hippocampal neurons. Rhodamine microspheres injected into the dorsal fornix clearly labelled the pyramidal cell layer of the hippocampus while excluding the other hippocampal layers and the dentate gyrus. In dissociated cell culture, the labelled cells were easily identified by fluorescence microscopy. Anti-neuron-specific enolase and anti-glial fibrillary acidic protein antibody staining confirmed that the labelled cells were neurons. The input resistance decreased from 2 GΩ to 450 MΩ, the input capacitance increased from 25 pF to 75 pF, the percentage of cells showing repetitive action potentials increased from 6% to 30%, and both peak GABA and glutamate responses increased over 100% during the 0 to 10 days time period investigated. This increase in chemosensitivity can be accounted for by an increase in cell size without an increase in the specific amino acid gated channel density. The subset of hippocampal neurons identified by the retrograde tracer technique are similar to non-labelled neurons with respect to the electrophysiological and pharmacological variables investigated. Nevertheless, it is likely that identified neurons may possess unique properties not evident in this study and refinement of the dissociated cell culture system using identified neuronal subpopulations may facilitate investigations looking at neuronal interactions such as synapse formation.     

Ethanol-induced death of postnatal hippocampal neurons

Fetal alcohol exposure causes severe neuropsychiatric problems, but mechanisms of the ethanol-associated changes in central nervous system development are unclear. In vivo, ethanol's interaction with N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid type A (GABA(A)) receptors may cause increased apoptosis in the immature forebrain. We examined whether ethanol affects survival of neonatal hippocampal neurons in primary cultures. A 6-day ethanol exposure killed hippocampal neurons with an LD50 of approximately 25 mM. Elevated extracellular potassium or insulin-related growth factor 1 inhibited cell loss. Although potentiation of GABA(A) receptors or complete block of NMDA receptors also kills hippocampal neurons, pharmacological studies suggest that ethanol's interaction with GABA(A) and NMDA receptors is not sufficient to explain ethanol's effects on neuronal survival. Ca(2+) influx in response to depolarization was depressed >50% by chronic ethanol treatment. We suggest that chronic ethanol may promote neuronal loss through a mechanism affecting Ca(2+) influx in addition to effects on postsynaptic GABA and glutamate receptors.     

Platelet-activating factor inhibits ionotropic GABA receptor activity in cultured hippocampal neurons

Platelet-activating factor (PAF), one of the most potent bioactive lipids, has been implicated in modulating long-term potentiation (LTP) and neurotoxicity. In the CNS, glutamate and GABA are the major excitatory and inhibitory neurotransmitters, respectively. Previous work has focused on the effects of PAF on glutamatergic receptor responses. The purpose of the present study was to investigate the possible actions of PAF on ionotropic GABA receptor responses in primary cultured hippocampal neurons using the whole-cell and single channel patch clamp techniques. Extracellular application of PAF induced a reduction of the GABA gated Cl- current in a majority of cells (29 of 44 cells), while it caused an enhancement in 10 of 44 cells. A similar heterogeneous modulation of PAF on the GABA receptor activities was also observed in outside-out patch recordings. Moreover, the cell-attached single channel recordings showed that PAF decreased the GABA channel activity. Therefore, PAF may modulate synaptic activity by inhibiting GABA receptor channels. During seizures and neural injury, when enhanced synthesis of this lipid mediator takes place, the actions of PAF on inhibitory GABA receptors may contribute to synaptic dysfunction.     

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).     

Ion channels activated by quinolinic acid in cultured rat hippocampal neurons

The membrane responses to quinolinic acid, an excitotoxic brain metabolite, were studied in cultured rat hippocampal neurons with the patch-clamp technique. In the whole-cell recording mode, pressure applications of quinolinic acid elicited inwardly directed membrane currents over a membrane potential range of −60 to −5 mV. The current response reversed at about 0 mV. The current-voltage (I–V) relation of the response had a negative slope conductance at membrane potentials more negative than −40 mV. On removal of Mg²⁺ from the extracellular solution, the current response showed no region of negative slope conductance at potentials more positive than −60 mV. In Mg²⁺-free solution applications of quinolinic acid elicited discrete pulse-like current flows through the outside-out membrane patch. The single channel conductance was 40–46 pS over a membrane potential range of −40 to −80 mV, and 50–55 pS at membrane potentials more positive than +30 mV, showing an outward rectification. These values of the single channel conductance were similar to those of the main conducting state of the channels activated by (NMDA). The responses to quinolinic acid were completely suppressed by the NMDA receptor antagonist (±)-2-amino-5-phosphonovaleric acid. The results indicate that quinolinic acid selectively activates NMDA receptors in the cultured rat hippocampal neurons.     

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.     

Testosterone attenuates beta-amyloid toxicity in cultured hippocampal neurons.

Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain's vulnerability to Alzheimer's disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult beta-amyloid in the presence of testosterone. beta-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.     

Glutamate-stimulated protein phosphorylation in cultured hippocampal pyramidal neurons.

The regulation of second-messenger production and protein phosphorylation by glutamate has been investigated in primary cultures of pure hippocampal pyramidal neurons. Embryonic rat pyramidal neurons were prepared according to the procedures of Bartlett and Banker (1984) and studied 1-21 d after plating. Glutamate caused a transient increase in intracellular free [Ca2+], determined with fura-2, in the presence of 1.26 mM extracellular Ca2+, but not in 50 nM free Ca(2+)-containing solution. Glutamate also transiently increased cellular diacylglycerol content in both normal and low-[Ca2+] media. Neurons were prelabeled with 32P-orthophosphate to label intracellular ATP, then stimulated with glutamate (100 microM). A rapid transient incorporation of 32P into primarily three proteins of 120, 87, and 48 kDa was found by analysis of two-dimensional gels. At 30 sec after glutamate stimulation, 32P incorporation into the 87-kDa and 48-kDa proteins peaked (240% and 170% basal levels, respectively), and by 2 min, phosphorylation of the 87-kDa protein had returned to basal levels, while that of the 48-kDa protein decreased but remained above control levels. The phosphorylation of these proteins appeared to be mediated by protein kinase C (PKC) because all three showed an increase in phosphorylation after phorbol ester treatment of cultures. Phosphate incorporation was accompanied by an acidic shift in the isoelectric point of both 87- and 48-kDa proteins. Glutamate stimulation resulted in phosphorylation in the presence and absence of Ca2+ influx. Antibody recognition and biochemical characteristics indicated that the 87-kDa phosphoprotein is the PKC substrate MARCKS (myristoylated, alanine-rich C-kinase substrate). The 48-kDa protein, though very similar to GAP-43, was not recognized by specific antibodies raised against GAP-43. These results suggest that glutamate stimulates the transient generation of second messengers that activate PKC in hippocampal neurons, resulting in a significant increase in the phosphorylation of three specific proteins.     

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.