Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Presynaptic k-Opioid and Muscarinic Receptors Inhibit the Calcium-Dependent Component of Evoked Glutamate Release from Striatal Synaptosomes

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.     

Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations

Intracellular free [Ca2+]i was measured using fura-2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L-(+)-Glutamate, D-(-)-glutamate, and quisqualate produced similar dose-dependent increases in [Ca2+]i, with EC50 values of 0.38 microM, 0.74 microM, and 0.1 microM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 microM) but had a greater maximum effect (57%). N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) did not increase [Ca2+]i. The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. L-2-Amino-4-phosphonobutyrate (L-AP4) (1 microM) completely blocked the changes in [Ca2+]i induced by L-(+)-glutamate, D-(-)-glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with L-(+)-serine-O-phosphate (L-SP) (1 mM) (which, like L-AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) had no effect on agonist-mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores.     

Metabotropic Glutamate Receptors Modulate Ischemia-Induced GABA Release in Mouse Hippocampal Slices

The involvement of glutamate receptors in GABA release in ischemia was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice. For in vitro ischemia, the slices were superfused in glucose-free media under nitrogen. Ionotropic glutamate receptor agonists failed to affect the ischemia-induced basal GABA release at either age. The K(+)-stimulated release in the immature hippocampus was potentiated by N-methyl-D-aspartate receptors, whereas in adults this release was reduced by both kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor activation. The group I metabotropic receptor agonist (1+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal ischemic GABA release in a receptor-mediated manner in adults, this being concordant with the positive modulation of GABAergic neurotransmission by group I metabotropic glutamate receptors. (1 +/-)-1-Aminocyclopentane-trans-1,3-dicarboxylate and (S)-3,5-dihydroxyphenylglycine also enhanced the K(+)-stimulated release in the developing hippocampus in a receptor-mediated manner. Because group I receptors generally increase neuronal excitability, the enhanced GABA release may attenuate hyperexcitation or strengthen inhibition, being thus neuroprotective, particularly under ischemic conditions. Group III metabotropic glutamate receptors were not at all involved in ischemic GABA release in the immature mice, but in adults their activation by O-phospho-L-serine potentiated the basal release and reduced the K(+)-stimulated release. These opposite effects were abolished by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Metabotropic glutamate receptors, namely group I and III receptors, are able to modify the release of GABA from hippocampal slices under ischemic conditions, both positive and negative effects being discernible, depending on the age and type of receptor activated.     

Group III Metabotropic Glutamate Receptors: Pharmacology,Physiology and Therapeutic Potential

Glutamate, the primary excitatory neurotransmitter in the central nervous system (CNS), exerts neuromodulatory actions via the activation of metabotropic glutamate (mGlu) receptors. There are eight known mGlu receptor subtypes (mGlu1-8), which are widely expressed throughout the brain, and are divided into three groups (I–III), based on signalling pathways and pharmacological profiles. Group III mGlu receptors (mGlu4/6/7/8) are primarily, although not exclusively, localised on presynaptic terminals, where they act as both auto- and hetero-receptors, inhibiting the release of neurotransmitter. Until recently, our understanding of the role of individual group III mGlu receptor subtypes was hindered by a lack of subtype-selective pharmacological tools. Recent advances in the development of both orthosteric and allosteric group III-targeting compounds, however, have prompted detailed investigations into the possible functional role of these receptors within the CNS, and revealed their involvement in a number of pathological conditions, such as epilepsy, anxiety and Parkinson’s disease. The heterogeneous expression of group III mGlu receptor subtypes throughout the brain, as well as their distinct distribution at glutamatergic and GABAergic synapses, makes them ideal targets for therapeutic intervention. This review summarises the advances in subtype-selective pharmacology, and discusses the individual roles of group III mGlu receptors in physiology, and their potential involvement in disease.     

Effect of Low pH on Glutamate Uptake and Release in Isolated Presynaptic Endings from Rat Brain

The effect of acidification of the incubation medium on the membrane potential and glutamate uptake and release was studied in isolated presynaptic neuronal endings (synaptosomes) from rat brain. Using the fluorescent probe diS-C₃-(5), a rapid depolarization of plasma membrane was detected at pH 6.0, most probably as a result of the inhibition of the sodium pump and potassium channel blockade. The membrane potential decrease did not result in increase of basal efflux of glutamate. Glutamate release following K⁺-induced depolarization was decreased upon lowering pH to 6.0. Acidosis inhibited mainly calcium-dependent (vesicular) release of glutamate and did not significantly reduce [¹⁴C]glutamate uptake. This inhibition of glutamate release but not of glutamate uptake may be a mechanism of the protective effect of acidosis during brain ischemia.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

NMDA and Non-NMDA Ionotropic Glutamate Receptors Modulate Striatal Acetylcholine Release via Pre- and Postsynaptic Mechanisms

Abstract: The effects of NMDA and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1–300 µ M ) facilitated acetylcholine release from slices in a dose-dependent manner. NMDA (100–300 µ M ) and AMPA (30–300 µ M ), however, subsequently inhibited acetylcholine release. NMDA (100 µ M )-induced facilitation was antagonized by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and dizocilpine (both 1–10 µ M ), whereas the 10 µ M AMPA effect was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1–30 µ M ). NMDA (100 µ M )-induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor l -nitroarginine (1–100 µ M ). Tetrodotoxin (0.5 µ M ) prevented the facilitatory effect of 3 µ M NMDA and AMPA, but left unchanged that of 30 µ M NMDA and 100 µ M AMPA. Acetylcholine release from synaptosomes was stimulated by KCI (7.5–100 m M ) in a dose-dependent manner. NMDA and AMPA maximally potentiated the 20 m M KCl effect at 1 µ M and 0.01 µ M , but were ineffective at 100 µ M and 10 µ M , respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 µ M NMDA and 0.01 µ M AMPA were antagonized by CPP (0.0001–1 µ M ) or dizocilpine (0.0001–10 µ M ) and by CNQX (0.001–1 µ M ), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre- and post-synaptic NMDA and non-NMDA ionotropic receptors.     

Glutamate,Glutamate Receptors,and Downstream Signaling Pathways

Glutamate is a nonessential amino acid, a major bioenergetic substrate for proliferating normal and neoplastic cells, and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways. Glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in chronic disabling brain disorders such as Schizophrenia and neurodegenerative diseases like Alzheimer''s, Parkinson''s, and multiple sclerosis. In this review, we discuss the structural and functional relationship of mGluRs and iGluRs and their downstream signaling pathways. The three groups of mGluRs, the associated second messenger systems, and subsequent activation of PI3K/Akt, MAPK, NFkB, PLC, and Ca/CaM signaling systems will be discussed in detail. The current state of human mGluR1a as one of the most important isoforms of Group I-mGluRs will be highlighted. The lack of studies on the human orthologues of mGluRs family will be outlined. We conclude that upon further study, human glutamate-initiated signaling pathways may provide novel therapeutic opportunities for a variety of non-malignant and malignant human diseases.     

Statistical Analysis of Membrane Potential Fluctuations: Relation with Presynaptic Spike Train

In a study of integration at the single neuron level, the relationships between the postsynaptic membrane potential and the presynaptic spike train were analyzed. Fluctuations in membrane potential of neurons in the visceral ganglion of Aplysia were measured and described by histograms. The histogram estimates the probability density function of the membrane potential. Comparisons were made among histograms when there was no synaptic input, and when there was a single input in which variations were made in the PSP (postsynaptic potential) sign, i.e. excitatory or inhibitory, and arrival statistics, e.g. slow or fast, regular, Poisson-like, or patterned. This was examined in cells where the membrane potential was constant and in cells in which there was spontaneous pacemaker activity. The form of the histogram depended on whether the neuron was spontaneously quiescent or a pacemaker, or whether it received presynaptic input and, if it did, on the sign and temporal characteristics of such input. From such histograms the mean firing rate of output spike trains can be predicted; additional information of a temporal nature is required, however, to predict features of the interval structure of the output train. Suggestions are made concerning the way the nervous system might utilize the information summarized in the membrane potential histogram.     

Involvement of Metabotropic Glutamate Receptors in Ischemia-Induced Taurine Release in the Developing and Adult Hippocampus

Metabotropic glutamate receptors have recently been envisaged as involved in both potentiation and prevention of ischemic and excitotoxic neuronal damage. The release of the inhibitory amino acid taurine is markedly enhanced in ischemia in both the immature and mature mouse hippocampus. The modulation of [³H]taurine release by metabotropic receptor agonists and antagonists was studied in hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. Agonists of group I, II and III metabotropic glutamate receptors generally reduced the ischemia-induced release in adult animals. In the immature hippocampus the group I agonists (S)-3,5-dihydroxyphenylglycine and (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate, which mainly enhance neuronal excitation, potentiated initial taurine release in ischemia. Ionotropic glutamate receptor agonists also enhance the ischemia-induced taurine release in developing mice. This glutamate-activated taurine release may thus constitute an important protective mechanism against excitotoxicity in the immature hippocampus.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of D-aspartate into guinea-pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+-independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+-independent net efflux of endogenous and labelled glutamate and aspartate in the absence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate-sensitive Na+-cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+-cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+-dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.