Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

鸡视网膜谷氨酸受体和GABA受体在两栖类卵母细胞中的表达

本工作利用两栖类卵母细胞作为功能表达系统,对鸡视网膜中的谷氨酸受体和ＧＡＢＡ受体的类型和基本性质进行了研究。在注射鸡视网膜ｍＲＮＡ的卵母细胞上,谷氨酸受体有明显的表达。Ｌ－Ｇｌｕ及其类似物ＫＡ,ＡＭＰＡ,ＱＡ都毫无例外地能诱导卵母细胞产生快速平滑的去极化电流,而ＮＭＤＡ,Ｌ－ＡＰ４,ＡＣＰＤ以及天冬氨酸不能诱导明显的电流反应。并且ＡＭＰＡ,ＱＡ对ＫＡ反应存在一定的抑制作用,提示ＡＭＰＡ,ＱＡ可能与ＫＡ作用于同一受体。抑制性氨基酸ＧＡＢＡ的受体被证明大部分为ＧＡＢＡＡ亚型,但有小部分的ＧＡＢＡ反应不能为荷包牡丹碱（ｂｉｃｕｃｕｌｌｉｎｅ）所阻断。     

In Vitro and In Vivo Pharmacology of trans- and cis-(±)-1-Amino-1,3-Cyclopentanedicarboxylic Acid: Dissociation of Metabotropic and Ionotropic Excitatory Amino Acid Receptor Effects

This study explored further the function of the metabotropic excitatory amino acid receptor in the rat brain. The trans and cis isomers of (+-)-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were characterized for relative affinities at ionotropic and metabotropic excitatory amino acid receptors in vitro, as well as ability to produce in vivo excitatory or excitotoxic effects in rats. trans-ACPD was about 12 times more potent in vitro as an agonist for metabotropic excitatory amino acid receptors when compared to its ability to displace N-methyl-D-aspartate (NMDA) ([3H]CGS-19755) receptor binding, cis-ACPD was about 30 times more potent as a displacer of [3H]CGS-19755 binding than as a stimulant of phosphoinositide hydrolysis. When administered intraperitoneally to neonatal rats, both cis- and trans-ACPD produced convulsions that were prevented by the competitive NMDA receptor antagonists, LY233053 and LY274614. cis-ACPD was six times more potent as a convulsant when compared to trans-ACPD. Both compounds were examined for excitotoxic effects in vivo following stereotaxic injection into the mature or neonatal rat striatum. Doses of trans-ACPD of up to 5,000 or 1,200 nmol produced few signs of striatal neuronal degeneration in the mature or neonatal brain, respectively. However, cis-ACPD produced extensive dose-related neuronal degeneration at doses of 100-1,000 nmol in the mature brain and 50-200 nmol in the neonatal brain. These studies suggest that, unlike the ionotropic excitatory amino acid receptors, activation of the metabotropic excitatory amino acid receptor does not result directly in excitatory effects, such as excitotoxicity.     

Desensitization of Metabotropic Glutamate Receptors in Neuronal Cultures

Preexposure of cultured cerebellar neurons to glutamate reduced the stimulation of polyphosphoinositide (PPI) hydrolysis induced by subsequent addition of glutamate without affecting the response to the muscarinic receptor agonist carbamylcholine. Desensitization of glutamate-stimulated PPI hydrolysis developed rapidly and persisted up to 48 h after removal of glutamate from the incubation medium. Stimulation of PPI hydrolysis by quisqualate was abolished in cultures pretreated with quisqualate or glutamate, but not with N-methyl-D-aspartate (NMDA). In contrast, pretreatment with NMDA reduced the stimulation of PPI hydrolysis induced by a subsequent addition of NMDA, leaving the action of quisqualate intact. The lack of cross-desensitization between NMDA and quisqualate supports the existence of two distinct subtypes of glutamate receptors coupled to PPI hydrolysis. Desensitization induced by a 30-min (but not by a 6-h) exposure to glutamate was attenuated or prevented by putative protein kinase C inhibitors, including mono- and trisialogangliosides, sphingosine, and polymyxin B, but not by inhibitors of arachidonic acid metabolism, nor by the nonselective calpain inhibitor leupeptin, nor by the lectin concanavalin A. These results suggest that desensitization of metabotropic glutamate receptors involves, at least in its rapid component, activation of protein kinase C.     

Carnitine Inhibits Hydrolysis of Inositol Phospholipids Induced by Activation of Metabotropic Receptors

We previously found that carnitine prevents glutamate neurotoxicity and that this effect is mediated by activation of metabotropic glutamate receptors. We show now that carnitine inhibits the hydrolysis of inositol phospholipids induced by different agonists of metabotropic glutamate receptors (tACPD; (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; DHPG, (R,S)-3,5-dyhydroxyphenylglycine or S4C3HPG, (S)-4-carboxy-3-hydroxyphenylglycine). The EC₅₀ was ca. 170 M and the inhibition was complete at 1 mM carnitine. Carnitine also inhibits completely hydrolysis of inositol phospholipids induced by arterenol (agonist of adrenoceptors) and only partially (ca. 50%) that induced by carbachol (agonist of muscarinic receptors). Carnitine did not inhibit phospholipase C activity but inhibits partially (43%) the hydrolysis of inositol phospholipids induced by direct activation of G proteins with AIP-₄. The results reported indicate that carnitine inhibits the hydrolysis of inositol phospholipids induced by activation of metabotropic receptors likely by interfering the function of some types of G proteins.     

Inhibition of Forskolin-Stimulated Cyclic AMP Formation by 1-Aminocyclopentane-trans-1,3-Dicarboxylate in Guinea-Pig Cerebral Cortical Slices

The effects of the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) on forskolin-stimulated cyclic AMP formation in guinea-pig cerebral cortex slices were determined. t-ACPD inhibited the accumulation of [3H]cyclic AMP by approximately 80%, with an IC50 value of 35 +/- 4 microM. The effect was reversible and stereoselective, with the 1S,3R isomer being approximately 400-fold more potent than the 1R,3S isomer. L-Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and N-methyl-D-aspartate (NMDA) were ineffective. The effect of t-ACPD was not blocked by antagonists of the phospholipase C-linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea-pig brain that is activated selectively by t-ACPD and that is negatively linked to adenylyl cyclase.     

Evidence for Metabotropic Excitatory Amino Acid Receptor Heterogeneity: Developmental and Brain Regional Studies

To examine whether multiple subtypes of the excitatory amino acid (EAA) receptor coupled to phosphoinositide (PPI) hydrolysis exist, we have pharmacologically characterized the PPI response in neonatal and adult rat brain. Activation of PPI hydrolysis was determined by the accumulation of [3H]inositol monophosphate in brain slices prelabeled with [3H]inositol. In neonatal hippocampus, D,L-2-amino-3-phosphonopropionic acid (AP3; 1 mM) inhibited the cis-1-aminocyclopentane-1,3-dicarboxylic acid (IUPAC nomenclature; ACPD; 100 microM)- and quisqualate (Quis; 100 microM)-stimulated PPI hydrolysis by 73 and 66%, respectively, but had no effect in neonatal cerebellum. In adult hippocampus, AP3 stimulated PPI hydrolysis with potency and efficacy comparable to those of Quis and ACPD and completely masked the Quis concentration-response curve. In adult cerebellum, only Quis behaved as a full agonist on the PPI response. The Quis concentration-response curve was shifted rightward with a fourfold decrease in potency in the presence of ACPD (5 mM), whereas it was nearly additive with the PPI response induced by AP3 (5 mM). Thus, our data reveal significant developmental and brain regional differences in metabotropic EAA receptor responses and support the notion that this receptor is heterogeneous, in both a regionally specific and a developmentally dependent manner.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 M) was abolished by 8-phenyltheophylline (15 uM). Glutamate and the glutamatergic agonists kainate or trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 M, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 M 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (rs)-alpha-methyl-4-carboxyphenylglycine [(rs)M-CPG], a metabotropic, antagonist, while 1 mMl-2-amino-3-phosphonopropionate (l-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 M and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.Abbreviations ADO adenosine - DNQX 6,7-dihydroxy-2,3-dinitro-quinoxaline - KA kainate - l-AP3 l-2-amino-3-phosphonopropionate - mGluRs metabotropic glutamate receptors - P-THEO 8-phenyltheophylline - (rs)M-CPG (rs)-alpha-methyl-4-carboxyphenyl-glycine - trans-ACPD trans-d,l-1-aminocyclopentane-1,3-dicarboxyho acid     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate in Cultured Radial Glia

The effect of L-glutamate (Glu) and its structural analogs N-methyl-D-aspartate (NMDA), kainate (KA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), on the activation of p42 mitogen activated protein kinase (MAPK) was examined in cultured chick radial glia cells, namely retinal Müller cells and cerebellar Bergmann cells. Glu, NMDA, AMPA and KA evoked a dose and time dependent increase in MAPK activity. AMPA and KA responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas NMDA responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate (CPP) indicating that the increase in MAPK activity is mediated by AMPA/low affinity KA and NMDA subtypes of Glu receptors. The present findings open the possibility of a MAPK cascade involvement in the regulation of Glu-induced gene expression in radial glia.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

Glutathione Is an Endogenous Ligand of Rat Brain N-Methyl-D-Aspartate (NMDA) and 2-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionate (AMPA) Receptors

A study was made of the effects of reduced (GSH) and oxidized (GSSG) glutathione on the Na⁺-independent and N-methyl-D-aspartate (NMDA) displaceable bindings of glutamate, on the binding of kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ligands of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (±)-3-(2-car-boxypiperazin-4-yl)propyl-1-phosphonate (CPP). GSH and GSSG strongly inhibited the binding of glutamate, CPP and AMPA, kainate and glycine binding being less affected. Both peptides enhanced the binding of dizocilpine in a time- and concentration-dependent manner. This activatory effect was not additive to that of saturating concentrations of glutamate or glutamate plus glycine. The activation of dizocilpine binding by GSH and GSSG was prevented by the competitive NMDA and glycine antagonists DL-2-amino-5-phosphonovalerate and 7-chlorokynurenate. GSH and GSSG may be endogenous ligands of AMPA and NMDA receptors, binding preferably to the glutamate recognition site via their -glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.     

Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation

The selective metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans-ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis-linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans-ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R- and 1S,3S-ACPD (but not 1R,3S-ACPD) induce a concentration-dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1S,3R-ACPD-stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.     

Glucose Regulates Glutamate-Evoked Arachidonic Acid Release from Cultured Striatal Neurons

Abstract: l -Glutamate stimulates the liberation of arachidonic acid from mouse striatal neurons via the activation of N -methyl- d -aspartic acid (NMDA) receptors and by the joint stimulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic receptors. In this study, we investigated whether starving cultured mouse striatal neurons of glucose would modify glutamatergic receptor-mediated arachidonic acid release. Glucose deprivation for 30 min led to enhancement of the NMDA-evoked release of arachidonic acid, compared with that observed in the presence of glucose. This enhanced response depended on both the concentration of glucose and the length of time of glucose deprivation. The enhanced NMDA response appeared to result from both a release of glutamate and the subsequent additional release of arachidonic acid due to the activation of AMPA and metabotropic receptors. Indeed, the increased NMDA response was completely reversed when extracellular glutamate was enzymatically removed. Moreover, glucose deprivation potentiated the combined AMPA/metabotropic receptor-evoked release of arachidonic acid, even in the absence of extracellular glutamate. However, removing glucose did not improve the calcium rise induced by AMPA or NMDA. The ATP-evoked release of arachidonic acid from striatal astrocytes was not altered by glucose starvation. In summary, glucose deprivation affected two properties of striatal neurons: (a) it induced an NMDA-evoked release of glutamate from striatal neurons and (b) it selectively potentiated the AMPA/(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid-evoked release of [³H]arachidonic acid without altering the authentic NMDA-mediated response.     

Non-Muscarinic- and Non-Adrenergic-Mediated Effects of Lindane on Phosphoinositide Hydrolysis in Rat Brain Cortex Slices

The influence of lindane upon phosphatidylinositol hydrolysis in rat brain cortex slices has been investigated using anion-exchange chromatography in order to separate the water-soluble inositol metabolites. Acetylcholine, noradrenaline, and lindane induce the accumulation of myo-[2-³H]inositol as the water-soluble inositol metabolites. However, the cholinergic muscarinic antagonist atropine inhibited the stimulatory response of carbachol, but practically unmodified the effect that lindane has on inositol phosphate production. Also, prazosin anti-₁ adrenoreceptors blocked noradrenaline-induced phosphoinositide hydrolysis, but had no effect on lindane-induced increase of inositol phosphate levels. The results suggest that lindane does not exert a general effect on the receptor-stimulated formation of inositol phosphates by both muscarinic and ₁-adrenergic agonists.     

Interactions Between Phospholipase C-Coupled and N-Methyl-D-Aspartate Receptors in Cultured Cerebellar Granule Cells: Protein Kinase C Mediated Inhibition of N-Methyl-D-Aspartate Responses

The N-methyl-D-aspartate (NMDA) receptor of rat cerebellar granule cells in primary culture is inhibited by phospholipase C-coupled receptor activation. In the absence of ionotropic agonist, cells modulate their cytoplasmic free Ca2+, [Ca2+]c, in response to stimulation of M3 muscarinic receptors, metabotropic glutamate receptors, and endothelin receptors by the respective agonists carbachol, trans-1-amino-1,3-cyclopentanedicarboxylic acid, and endothelin-1. The response is consistent with the ability of phospholipase C-coupled receptors to release a pool of intracellular Ca2+ and induce a subsequent Ca2+ entry into the cell; both of these responses can be abolished by discharge of internal Ca2+ stores with low concentrations of ionomycin or thapsigargin. In the case of cells stimulated with NMDA, the [Ca2+]c response to the phospholipase C-coupled agonists is complex and agonist dependent; however, in the presence of ionomycin each agonist produces a partial inhibition of the NMDA component of the [Ca2+]c signal. This inhibition can be mimicked by the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate. It is concluded that NMDA receptors on cerebellar granule cells are inhibited by phospholipase C-coupled muscarinic M3, glutamatergic, and endothelin receptors via activation of protein kinase C.     

脑外伤时猫大脑皮质和海马N─甲基─D─天冬氨酸受体的变化

本实验采用放射性配基受体结合分析法,测定了猫脑外伤时大脑皮质和海马Ｎ─甲基─Ｄ─天冬氨酸受体（ＮＭＤＡＲ）的变化。并用氨基酸自动分析仪观察了猫大脑皮质兴奋性氨基酸含量变化。结果表明,伤后２和６ｈ两侧大脑皮质和海马ＮＭＤＡＲ的最大结合容量明显降低,伤后２ｈ以海马变化最大,并以伤后６ｈ伤侧大脑皮质中降低最为显著;而大脑皮质兴奋性氨基酸含量伤后５ｍｉｎ即显著升高,然后呈下降趋势,且以伤侧大脑皮质变化为大。提示：脑外伤后ＮＭＤＡＲ的下调与兴奋性氨基酸的大量释放有关,可能在兴奋毒性脑损伤中起重要作用。     

Enhanced Sensitivity of "Metabotropic" Glutamate Receptors After Induction of Long-Term Potentiation in Rat Hippocampus

Stimulation of [3H]inositol monophosphate ([3H]InsP) formation by ibotenate or trans-1-aminocyclopentyl-1,3-dicarboxylic acid (t-ACPD) in rat hippocampal slices was enhanced after tetanic stimulation of the Schaffer collaterals projecting to the CA1 region (in vitro) or the perforant pathway projecting to the dentate gyrus (in freely moving animals). This effect was observed 5 h (but not 2 h) after long-term potentiation (LTP) induction and was abolished if tetanic stimulation was performed in the presence of specific antagonists of N-methyl-D-aspartate receptors. The delayed increase in excitatory amino acid-induced polyphosphoinositide (PPI) hydrolysis was accompanied by an enhanced responsiveness to norepinephrine, whereas the basal and carbamylcholine-stimulated [3H]InsP formation were unchanged. These results suggest that an increased activity of "metabotropic" glutamate receptors may contribute to the synaptic mechanisms enabling the late expression and or maintenance of LTP. Accordingly, LTP decayed more rapidly (within 5 h) in rats repeatedly injected with LiCl (60-120 mg/kg, i.p., for 10 days), a treatment that led to a reduced efficacy of ibotenate and norepinephrine in stimulating PPI hydrolysis in hippocampal slices.     

The involvement of NMDA and AMPA receptors in the mechanism of antidepressant-like action of zinc in the forced swim test

Antidepressant-like activity of zinc in the forced swim test (FST) was demonstrated previously. Enhancement of such activity by joint administration of zinc and antidepressants was also shown. However, mechanisms involved in this activity have not yet been established. The present study examined the involvement of the NMDA and AMPA receptors in zinc activity in the FST in mice and rats. Additionally, the influence of zinc on both glutamate and aspartate release in the rat brain was also determined. Zinc-induced antidepressant-like activity in the FST in both mice and rats was antagonized by N-methyl-d-aspartic acid (NMDA, 75 mg/kg, i.p.) administration. Moreover, low and ineffective doses of NMDA antagonists (CGP 37849, L-701,324, d-cycloserine, and MK-801) administered together with ineffective doses of zinc exhibit a significant reduction of immobility time in the FST. Additionally, we have demonstrated the reduction of immobility time by AMPA receptor potentiator, CX 614. The antidepressant-like activity of both CX 614 and zinc in the FST was abolished by NBQX (an antagonist of AMPA receptor, 10 mg/kg, i.p.), while the combined treatment of sub-effective doses of zinc and CX 614 significantly reduces the immobility time in the FST. The present study also demonstrated that zinc administration potentiated a veratridine-evoked glutamate and aspartate release in the rat’s prefrontal cortex and hippocampus. The present study further suggests the antidepressant properties of zinc and indicates the involvement of the NMDA and AMPA glutamatergic receptors in this activity.     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Carbachol-Induced Hydrolysis of Phospholipids in Hippocampal Slices May Be Mediated in Part By Subsequent Activation of Metabotropic Glutamate Receptors

We observed that AP-3, an antagonist of metabotropic glutamate receptors, reduced carbachol-induced hydrolysis of phospholipids in hippocampal slices. This inhibition could be explained in different ways, e.g.: 1) AP-3 acts also as antagonist of muscarinic receptors mediating the hydrolysis of phospholipids induced by carbachol, 2) Carbachol induces the release of glutamate which, by activating metabotropic glutamate receptors, leads to additional hydrolysis of phospholipids. The aim of this work was to test these possibilities. It is shown that AP-3 reduces carbachol-induced hydrolysis of phospholipids in hippocampal slices but not in cerebellar neurons at 10–14 days of culture, when these cells are not able to induce hydrolysis of phospholipids following activation of metabotropic glutamate receptors. It is also shown that carbachol induces a release of [³H]aspartate in hippocampal slices. The results reported suggest that the hydrolysis of phospholipids induced by carbachol in hippocampal slices would have two components. One part would be due to direct activation by carbachol of muscarinic receptors associated to activation of phospholipase C. This part would not be inhibited by AP-3. The second part would be due to subsequent release of glutamate and activation of metabotropic glutamate receptors. This part would be inhibited by AP-3.