(1S,3R)-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Induced Increases in Cyclic AMP Formation in the Neonatal Rat Hippocampus Are Mediated by a Synergistic Interaction Between Phosphoinositide- and Inhibitory Cyclic AMP-Coupled mGluRs

Abstract: A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1 S ,3 R )-1-Aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), which is an agonist for phosphoinositide-coupled and inhibitory-coupled cAMP-linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five- to 10-fold). However, the agonists 3,5-dihydroxyphenylglycine (DHPG) and (2 R ,4 R )-4-aminopyrrolidine-2,4-dicarboxylate (2 R ,4 R -APDC), which selectively act on phosphoinositide-coupled and inhibitory cAMP-coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype-selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1 S ,3 R -ACPD, were found. Stimulations of cAMP content evoked by 1 S ,3 R -ACPD and combined additions of DHPG plus 2 R ,4 R -APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8- p -sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)-α-methyl-4-carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1 S ,3 R -ACPD-induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).     

4-Bromohomoibotenic Acid Selectively Activates a 1-Aminocyclopentane-1S,3R-Dicarboxylic Acid-Insensitive Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis in Rat Cortical Slices

Abstract: Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue ( R,S )-4-bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration-dependent manner (EC₅₀ = 190 µ M ). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1 S ,3 R -ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to l -2-amino-3-phosphonopropionic acid ( l -AP3), whereas the response to 1 S ,3 R -ACPD is partially blocked by l -AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis-linked mGluR that is insensitive to 1 S ,3 R -ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.     

3,5-Dihydroxyphenylglycine Is a Highly Selective Agonist for Phosphoinositide-Linked Metabotropic Glutamate Receptors in the Rat Hippocampus

Abstract: Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein-coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid and (2 S ,3 S ,4 S )α-(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin-stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5-dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin-stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist-mediated increases or decreases in cAMP formation are not secondary to mGluR-mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5-dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP-mGluR second messenger systems.     

The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

Activation of Metabotropic Glutamate Receptors Prevents Neuronal Apoptosis in Culture

Abstract: Cultured granule cells grown in serum-containing medium with a "low K⁺" concentration (10 m M ) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) prevented the development of low K⁺-induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1 S ,3 R -ACPD was prevented by the mGluR antagonist ( RS )-α-methyl-4-carboxyphenylglycine (MCPG) and was mimicked by N -methyl- d -aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li⁺—which reduced polyphosphoinositide response to concentrations of glutamate (5 µ M ) that approximate those physiologically present in the incubation medium—or MCPG, the development of low K⁺-induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against apoptosis during early stages of maturation.     

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes

We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD-catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi-prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR-mediated phosphoinositide phospholipase activation [quisqualate > (S)-3,5-dihydroxyphenylglycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid]. The response to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was inhibited by the mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and, less potently, by 1-aminoindan-1,5-dicarboxylic acid and 4-carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.     

N-Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells

Abstract: In previous studies, we demonstrated that the neuropeptide, N -acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1–6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans -1-aminocyclopentane-1,3-dicarboxylate ( trans -ACPD) and l -2-amino-4-phosphonobutyrate ( l -AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans -ACPD, activated each of the transfected receptors, whereas l -AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans -ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

Phosphorylation of Mitogen-Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylate and the mGluR group I-selective agonists ( RS )-3,5-dihydroxyphenylglycine (DHPG) and l -quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist l (+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.     

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, 1R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino-3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs.     

Long-term depression of mGluR1 signaling

Glutamate produces both fast excitation through activation of ionotropic receptors and slower actions through metabotropic receptors (mGluRs). To date, ionotropic but not metabotropic neurotransmission has been shown to undergo long-term synaptic potentiation and depression. Burst stimulation of parallel fibers releases glutamate, which activates perisynaptic mGluR1 in the dendritic spines of cerebellar Purkinje cells. Here, we show that the mGluR1-dependent slow EPSC and its coincident Ca transient were selectively and persistently depressed by repeated climbing fiber-evoked depolarization of Purkinje cells in brain slices. LTD(mGluR1) was also observed when slow synaptic current was evoked by exogenous application of a group I mGluR agonist, implying a postsynaptic expression mechanism. Ca imaging further revealed that LTD(mGluR1) was expressed as coincident attenuation of both limbs of mGluR1 signaling: the slow EPSC and PLC/IP3-mediated dendritic Ca mobilization. Thus, different patterns of neural activity can evoke LTD of either fast ionotropic or slow mGluR1-mediated synaptic signaling.     

Metabotropic glutamate receptor activation and intracellular cyclic ADP-ribose release Ca2+ from the same store in cultured DRG neurones

The whole cell patch clamp technique has been used to record Ca(2+)-activated cation and chloride conductances evoked by release of Ca2+ from intracellular stores of cultured neonatal dorsal root ganglion neurones. The aim of this study was to investigate metabotropic glutamate receptor (mGluR) mechanisms and evaluate a possible role for cyclic ADP-ribose as an intracellular signalling molecule. Glutamate and the metabotropic glutamate receptor agonist (1S, 3R)-ACPD-evoked transient depolarizations, Ca(2+)-activated inward currents and rises in intracellular Ca2+. The (1S, 3R)-ACPD-activated currents were insensitive to InsP3 signalling inhibitors, heparin and pentosan polysulphate. Intracellular application of ryanodine alone activated currents in this study and proved a difficult tool to use as a potential inhibitor of cyclic ADP-ribose-mediated responses. However, intracellular dantrolene did attenuate both (1S, 3R)-ACPD and cyclic ADP-ribose responses. Intracellular photo-release of cGMP and cyclic ADP-ribose mimicked the responses to mGluR receptor activation. Intracellular application of nicotinamide and W7 inhibited the responses to photo-released cGMP but did not prevent responses to mGluR activation. The cyclic ADP-ribose receptor antagonist 8-amino cyclic ADP-ribose attenuated responses to (1S, 3R)-ACPD, cGMP and cyclic ADP-ribose, but some Ca(2+)-activated inward currents were still observed in the presence of this antagonist. In conclusion, mGluR receptor activation, cGMP and cyclic ADP-ribose release Ca2+ from intracellular stores. Some evidence suggests that pharmacologically related pathways are involved.     

Depression of excitatory transmission at PF-PC synapse by group III metabotropic glutamate receptors is provided exclusively by mGluR4 in the rodent cerebellar cortex

In the rodent cerebellum, pharmacological activation of group III pre-synaptic metabotropic glutamate receptors (mGluRs) by the broad spectrum agonist l -2-amino-4-phosphonobutyric acid, acutely depresses excitatory synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses. Among the group III mGluR subtypes, cerebellar granule cells express predominantly mGluR4, but also mGluR7 and mGluR8 mRNA. Taking into account that previous functional and pharmacological studies have used group III mGluR broad spectrum agonists that do not differentiate between these various subtypes, their relative contribution to the modulation of glutamatergic transmission at PF-PC synapses remains to be elucidated. In order to clarify this issue, we applied conventional whole-cell patch-clamp recordings and pre-synaptic calcium influx measurements, combined with pharmacological manipulations to rat and mice cerebellar slices. With the use of (1 S ,2 R )-1-amino-2-phosphonomethylcyclopropanecarboxylic acid, a new and selective group III mGluR agonist, N -phenyl-7-(hydroxylimino)cyclopropa[b]-chromen-1a-carboxamide, the specific positive allosteric modulator of mGluR4, ( S )-3,4-dicarboxyphenylglycine, a selective mGluR8 agonist, and mGluR4 knock-out mice, we demonstrate that the inhibitory control of group III mGluRs on excitatory neurotransmission at PF-PC synapses of the rodent cerebellar cortex, is totally because of the activation of pre-synaptic mGluR4 autoreceptors.     

Endogenous Adenosine Regulates the Apparent Efficacy of 1-Aminocyclopentyl-1S,3R-Dicarboxylate Inhibition of Forskolin-Stimulated Cyclic AMP Accumulation in Rat Cerebral Cortical Slices

Abstract: In rat cerebral cortical slices, the 1-aminocyclopentyl-1 S ,3 R -dicarboxylate (1 S ,3 R -ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in a concentration-dependent manner with a maximal inhibition of 51 ± 3% and a half-maximally effective concentration of 8.8 ± 3.4 μ M . Similarly, 1 R ,3 S -ACPD inhibited the forskolin response in a concentration-dependent manner, but with an inhibition of 80 ± 5% at 3 μ M . In addition to inhibiting forskolin-stimulated cAMP levels, 1 S ,3 R -ACPD, but not 1 R ,3 S -ACPD, enhanced the cAMP response to A_2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1 S ,3 R -ACPD was increased (88 ± 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine also increased the inhibitory effect of 100 μ M 1 S ,3 R -ACPD, from 57 ± 1 to 78 ± 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1 S ,3 R -ACPD inhibition of forskolin-stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.     

Glutamate Exocytosis and MARCKS Phosphorylation Are Enhanced by a Metabotropic Glutamate Receptor Coupled to a Protein Kinase C Synergistically Activated by Diacylglycerol and Arachidonic Acid

Abstract: 4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K⁺ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4β-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )-ACPD] mimics the action of 4β-phorbol dibutyrate, but only in the presence of 2 µ M arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1 S ,3 R )-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca²⁺ channel or Ca²⁺-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1 S ,3 R )-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1 S ,3 R )-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.     

Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase a(2) alpha regulate arteriole responses in mouse neocortical brain slices

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-) (/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.     

Temporal and Depolarization-Induced Changes in the Absolute Amounts of mRNAs Encoding Metabotropic Glutamate Receptors in Cerebellar Granule Neurons In Vitro

Abstract: Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3–13 days in the presence of either 10 or 25 m M KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 m M than in 25 m M KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 m M KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 m M KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.     

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up-regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca²⁺]_i) in cultured rat astrocytes. Single-cell [Ca²⁺]_i recordings indicated that an mGluR-selective agonist, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate (1 S ,3 R -ACPD), elicits [Ca²⁺]_i oscillations in good agreement with the growth factor-induced up-regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1 S ,3 R -ACPD-mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12-myristate 13-acetate completely abolished the [Ca²⁺]_i increase. These and other pharmacological properties of 1 S ,3 R -ACPD-induced [Ca²⁺]_i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca²⁺]_i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca²⁺]_i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca²⁺]_i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.     

Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R-Aminocyclopentane-1,3-Dicarboxylic Acid in Brain Slices

Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.