Modulation of excitatory synaptic transmission in locus coeruleus by multiple presynaptic metabotropic glutamate receptors

Metabotropic glutamate receptors have been implicated in modulation of synaptic transmission in many different systems. This study reports the effects of selective activation of metabotropic glutamate receptors on synaptic transmission in intracellularly recorded locus coeruleus neurons in brain slice preparations. Perfusion of either L-2-amino-4-phosphonobutyric acid (L-AP4; 0.1-500 microM) or (+/-)-1-aminocyclopentane-trans-1,3,dicarboxylic acid (t-ACPD; 0.1-500 microM) caused a depression of excitatory postsynaptic potentials in a dose-dependent fashion to about 70% inhibition. Both agonists exerted their effects at relatively low concentrations with estimated EC50s of 2.6 microM and 11.5 microM for L-AP4 and t-ACPD, respectively. This inhibition was not observed with the potent group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 microM). Conversely, (R)-4-carboxy-3-hydroxyphenyl-glycine (4C-3H-PG), a group I antagonist/group II agonist, and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a novel and specific group II agonist, also caused an inhibition of excitatory postsynaptic potentials. Both t-ACPD and L-AP4 produced an increase in paired-pulse facilitation, and failed to change the locus coeruleus response to focally applied glutamate, indicating a presynaptic locus of action. The L-AP4 inhibition was antagonized by (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4: group III antagonist) but not by (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG; mixed antagonist], suggesting that this agonist acts through a type 4 metabotropic glutamate receptor. Conversely, t-ACPD was antagonized by MCPG and by ethyl glutamate (group II antagonist), but not by aminoindan dicarboxylic acid (AIDA; group I antagonist) or MAP4, suggesting that this agonist acts on a type 2 or 3 metabotropic glutamate receptor. Taken together, these results suggest that two pharmacologically distinct presynaptic metabotropic glutamate receptors function in an additive fashion to inhibit excitatory synaptic transmission in locus coeruleus neurons. These receptors may be involved in a feedback mechanism and as such may function as autoreceptors for excitatory amino acids.     

Metabotropic glutamate receptors in the rat nucleus accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCG1 > L-AP4 > (1S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca2+ currents through N- or P-type Ca2+ channels, as inhibition persisted in the presence of omega-conotoxin-GVIA or omega-Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (R,S)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I(AHP)). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

The role of nigrostriatal dopamine in metabotropic glutamate agonist-induced rotation

Metabotropic glutamate receptors are a major class of excitatory amino acid receptors. Eight metabotropic glutamate receptors subtypes have been cloned and have been classified into three groups based on their amino acid sequence homology, effector systems, and pharmacological profile. Previous results have shown that striatal group I metabotropic glutamate receptor stimulation produces vigorous contralateral rotation in intact rats, thought to be due to increased striatal dopamine release. Examination of FOS-like immunoreactivity and local cerebral glucose metabolism suggests that this occurs secondary to activation of the subthalamic nucleus. We sought to determine the contribution of dopamine by examining metabotropic glutamate receptor agonist-induced rotation in rats following acute dopamine depletion by reserpine/alpha-methyl-para-tyrosine treatment, or chronic dopamine depletion by 6-hydroxydopamine treatment. In unilateral 6-hydroxydopamine lesioned rats, the group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine induced contralateral rotation with a coincident increase in striatal 3,4-dihydroxyphenylacetic acid. The rotation was attenuated by the group I antagonist 1-aminoindan-1,5-dicarboxylate. Examination of FOS-like immunoreactivity and [14C]2-deoxyglucose uptake in chronically dopamine depleted rats also revealed similar patterns to those seen previously in intact rats. However, acutely dopamine depleted rats do not exhibit metabotropic glutamate receptor agonist-induced rotation and show a different pattern of [14C]2-deoxyglucose uptake, with no increase in glucose utilization in the intermediate and deep layers of the superior colliculus. These results suggest that there are compensatory changes under conditions of chronic dopamine denervation which permit metabotropic glutamate receptor agonist-induced rotation to occur, which may include dopamine receptor supersensitivity, increased dopamine turnover, and/or changes in sensitivity of striatal group I metabotropic glutamate receptors. The group III metabotropic glutamate receptor agonist L-(+)-2-amino-4-phosphonobutyrate induced contralateral rotation in 6-hydroxydopamine lesioned rats, while it had no effect in intact rats. Additionally, examination of FOS-like immunoreactivity revealed a distinct pattern following L-(+)-2-amino-4-phosphonobutyrate administration in 6-hydroxydopamine lesioned versus intact rats. These results suggest that there is a change in the effect of striatal group III stimulation under conditions of dopamine depletion.     

Pharmacological characterization of desensitization in a human mGlu1 alpha-expressing non-neuronal cell line co-transfected with a glutamate transporter

1. Stimulation of phosphoinositide hydrolysis by human mGlu1 alpha (HmGlu1 alpha) was examined in a non-neuronal cell line (AV12-664) co-expressing both HmGlu1 alpha and a rat glutamate/aspartate transporter (GLAST). 2. Desensitization of HmGlu1 alpha could be elicited by inhibition of the GLAST transporter with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). Maximal inhibition of HmGlu1 alpha-mediated phosphoinositide hydrolysis was induced upon 24 h pretreatment with trans-PDC. The concentration of glutamate in the extracellular medium also rose significantly in cells pretreated with trans-PDC. Glutamate levels increased upon incubation with trans-PDC in a time-dependent manner, with maximal glutamate levels attained after 24 h incubation with trans-PDC. 3. The time required for desensitization of HmGlu1 alpha by trans-PDC was compared to the time course for desensitization elicited by the direct-acting mGlu receptor agonists, 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) and (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG). Both direct-acting mGlu receptor agonists elicited desensitization of HmGlu1 alpha more rapidly than did trans-PDC, with maximal inhibition of agonist-induced phosphoinositide hydrolysis upon 12 h pretreatment. Agonist-induced desensitization could be fully reversed upon washout of agonist for 12 h. 4. Both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha could be blocked by inclusion of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an mGlu receptor antagonist, in the pretreatment medium. 5. Agonist-stimulated phosphoinositide hydrolysis by HmGlu1 alpha was found to parallel closely agonist-induced desensitization of HmGlu1 alpha. Thus, the EC50 values for 1S,3R-ACPD- and 3,5-DHPG-stimulated phosphoinositide hydrolysis were similar to the EC50 values for eliciting desensitization of HmGlu1 alpha. 6. These studies demonstrate desensitization of recombinant human mGlu1 alpha receptor in a non-neuronal cell line in which the receptor can be regulated by direct activation or by manipulation of glutamate transporter activity. Desensitization of HmGlu1 alpha was found to be mediated by activation of the receptor since the mGlu receptor antagonist, MCPG, blocked both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha. Furthermore, agonist-induced desensitization of HmGlu1 alpha was found to parallel receptor-mediated stimulation of phosphoinositide hydrolysis.     

Modulation of multiple potassium currents by metabotropic glutamate receptors in neurons of the hypothalamic supraoptic nucleus

We studied the effects of activation of the metabotropic glutamate receptors on intrinsic currents of magnocellular n urons of the supraoptic nucleus (SON) with whole cell patch-clamp and conventional intracellular recordings in coronal slices (400 micron) of the rat hypothalamus. Trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 10-100 microM), a broad-spectrum metabotropic glutamate receptor agonist, evoked an inward current (18.7 +/- 3.45 pA) or a slow depolarization (7.35 +/- 4.73 mV) and a 10-30% decrease in whole cell conductance in approximately 50% of the magnocellular neurons recorded at resting membrane potential. The decrease in conductance and the inward current were caused largely by the attenuation of a resting potassium conductance because they were reduced by the replacement of intracellular potassium with an equimolar concentration of cesium or by the addition of potassium channel blockers to the extracellular medium. In some cells, trans-ACPD still elicited a small inward current after blockade of potassium currents, which was abolished by the calcium channel blocker, CdCl2. Trans-ACPD also reduced voltage-gated and Ca2+-activated K+ currents in these cells. Trans-ACPD reduced the transient outward current (IA) by 20-70% and/or the IA-mediated delay to spike generation in approximately 60% of magnocellular neurons tested. The cells that showed a reduction of IA generally also showed a 20-60% reduction in a voltage-gated, sustained outward current. Finally, trans-ACPD attenuated the Ca2+-dependent outward current responsible for the afterhyperpolarization (IAHP) in approximately 60% of cells tested. This often revealed an underlying inward current thought to be responsible for the depolarizing afterpotential seen in some magnocellular neurons. (RS)-3,5-dihydroxyphenylglycine, a group I receptor-selective agonist, mimicked the effects of trans-ACPD on the resting and voltage-gated K+ currents. (RS)-alpha-methyl-4-carboxyphenylglycine, a group I/II metabotropic glutamate receptor antagonist, blocked these effects. A group II receptor agonist, 2S,1'S,2'S-2carboxycyclopropylglycine and a group III receptor agonist, (+)-2-amino-4-phosphonobutyric acid, had no effect on the resting or voltage-gated K+ currents, indicating that the reduction of K+ currents was mediated by group I receptors. About 80% of the SON cells that were labeled immunohistochemically for vasopressin responded to metabotropic glutamate receptor activation, whereas only 33% of labeled oxytocin cells responded, suggesting that metabotropic receptors are expressed preferentially in vasopressinergic neurons. These data indicate that activation of the group I metabotropic glutamate receptors leads to an increase in the postsynaptic excitability of magnocellular neurons by blocking resting K+ currents as well as by reducing voltage-gated and Ca2+-activated K+ currents.     

Modulation of pre- and postsynaptic calcium dynamics by ionotropic glutamate receptors at a plastic synapse

This study was conducted to assess the role of ionotropic glutamate receptors in the modulation of calcium dynamics on both sides of a vertebrate plastic synapse. Retrograde labeling of neuronal elements with high-affinity calcium-sensitive dyes was used in conjunction with confocal imaging techniques in an in vitro lamprey brain stem preparation. A prolonged calcium transient was measured both pre- and postsynaptically in response to a period of high-frequency ("tetanic") stimulation to the vestibulospinal-reticulospinal synapse. The ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and D,L-2-amino-5-phosphonopentanoate (D,L-AP5; 100 microM) reduced the calcium signal in both compartments of the synapse. The presynaptic D,L-AP5-sensitive component was enhanced markedly by the removal of Mg2+ from the superfusate. Increasing the extracellular stimulus intensity progressively augmented the presynaptic calcium signal, suggesting the recruitment of excitatory axo-axonic inputs onto these fibers. Further, the presence of an excitatory amino acid-mediated presynaptic potential underlying a component of the Ca2+ signal was demonstrated by electrophysiological recordings from vestibulospinal axons. Bath application of agonist, in the presence of tetrodotoxin (1 microM), confirmed the existence of N-methyl-D-aspartate receptors at the presynaptic element capable of modulating calcium levels. The postsynaptic Ca2+ response, which is known to be necessary for long-term potentiation (LTP) induction at this synapse, was localized to areas of the dendritic tree that correlated with the location of known synaptic inputs; thus the synaptically activated rise in postsynaptic calcium may confer the synapse specificity of LTP induction previously demonstrated. In summary, we have demonstrated the existence of physiologically activated presynaptic ionotropic glutamate receptors that are capable of modulating levels of intracellular calcium and have highlighted the importance of receptor-mediated increases in postsynaptic calcium for neuronal plasticity in the lamprey.     

Regulation of intrinsic and synaptic properties of neonatal rat trigeminal motoneurons by metabotropic glutamate receptors

We studied how metabotropic glutamate receptor (mGluR) activation modifies the synaptic and intrinsic membrane properties of neonatal rat trigeminal motoneurons using the broad-spectrum mGluR agonist (1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid [(1S,3R)-ACPD], group I/II antagonist (+/-)-alpha-methyl-4-carboxy-phenylglycine (MCPG), and group III agonist L-2-amino-4-phosphonobutanoate (L-AP4). (1S,3R)-ACPD depressed excitatory transmission to trigeminal motoneurons presynaptically and postsynaptically via presynaptic inhibition and by reducing the currents carried by ionotropic glutamate receptors selective for AMPA. (1S,3R)-ACPD also depolarized trigeminal motoneurons and increased input resistance by suppressing a Ba2+-sensitive leakage K+ current. These effects were not mimicked by L-AP4 (100-200 microM). High-threshold Ca2+ currents were also suppressed by (1S,3R)-ACPD. Repetitive stimulation of excitatory premotoneurons mimicked the postsynaptic effects of (1S, 3R)-ACPD. The postsynaptic effects of (1S,3R)-ACPD and repetitive stimulation were both antagonized by MCPG, suggesting that mGluRs were similarly activated in both experiments. We conclude that mGluRs can be recruited endogenously by glutamatergic premotoneurons and that mGluR-mediated depression of excitatory transmission, combined with increased postsynaptic excitability, enhances the signal-to-noise ratio of oral-related synaptic input to trigeminal motoneurons during rhythmical jaw movements.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [3H]cyclohexyladenosine (CHA) to the cellular fractions and P2 subfractions of the goldfish brain was studied. The A1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P2), 30 mM K+ stimulated glutamate, taurine and GABA release in a Ca(2+)-dependent fashion, whereas the aspartate release was Ca(2+)-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 microM) inhibited K(+)-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K+ (30 mM) stimulated Ca2+ influx (46.8 +/- 6.8%) and this increase was completely abolished by pretreatment with 100 nM omega-conotoxin. Pretreatment with 100 microM R-PIA or 100 microM CHA, reduced the evoked increase of intra-synaptosomal Ca2+ concentration, respectively by 37.7 +/- 4.3% and 39.7 +/- 9.0%. A possible correlation between presynaptic A1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus

The group I specific metabotropic glutamate (mGlu) receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) (100 microM, 10 min) induced long-term depression (LTD) of synaptic transmission in the CA1 region of adult rat hippocampal slices, measured using a grease-gap recording technique. In "normal" (1 mM Mg2+-containing) medium, LTD (measured 30 min after washout of DHPG) was small (13+/-3%), but LTD was enhanced if DHPG was applied when the tissue was made hyperexcitable, either by omitting Mg2+ from the perfusate (35+/-3%) or by adding the GABA(A) receptor antagonist picrotoxin (29+/-2%). The N-methyl-D-aspartate (NMDA) receptor antagonist AP5 (100 microM) substantially reduced the generation of DHPG-induced LTD in Mg2+-free medium, but had little effect on LTD induced in the presence of picrotoxin. In Mg2+-free medium, the threshold concentration of DHPG required to induce LTD was between 1 and 3 microM. Neither agonists specific for group II (100 nM DCG-IV or 1 microM LY354740) or group III (10 microM L-AP4) mGlu receptors or a combined group I and II agonist (30-100 microM (1S,3R)-ACPD) induced LTD. However, an agonist (1 mM CHPG) which activates mGlu5 but not mGlu1 receptors did induce LTD. Surprisingly, DHPG-induced LTD was reversed by mGlu receptor antagonists, applied hours after washout of DHPG. DHPG-induced LTD did not occlude with LTD induced by synaptic activation (1200 stimuli delivered at 2 Hz), in Mg2+-free medium. These data show that activation of group I mGlu receptors (probably mGlu5) can induce LTD and that this mGlu receptor-mediated LTD may, or may not, require activation of NMDA receptors, depending on the experimental conditions.     

Group I metabotropic glutamate receptors mediate slow inhibition of calcium current in neocortical neurons

Metabotropic glutamate receptor (mGluR)-mediated inhibition of high-voltage-activated Ca2+ currents was investigated in pyramidal neurons acutely isolated from rat dorsal frontoparietal neocortex. Whole cell recordings were made at 30-32 degrees C, with Ca2+ as the charge carrier. Selective agonists were used to classify the subgroup of mGluRs mediating the response. Ca2+ currents were inhibited by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and by the group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) but not by the group II agonist (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV) or the group III agonist (+)-2-amino-4-phosphonobutryic acid (-AP4). (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (-CCG-I) was effective at 10 and 100 microM but not at 1 microM, consistent with involvement of group I mGluRs. Variable results were obtained with the putative mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) and the putative mGluR1-selective antagonist (S)-4-carboxyphenylglycine [(S)-4CPG], indicating that the group I mGluR subtypes may vary between cells or that these compounds were activating other receptors. The actions of (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG] were consistent with it being a low-potency antagonist. Several features of the Ca2+ current inhibition evoked by DHPG distinguished it from the rapid modulation typical of a direct action of G proteins on Ca2+ channels; the inhibition was slow to reach maximum (tens of seconds), current activation was not slowed or shifted in the positive voltage direction, and the inhibition was not relieved by positive prepulses. Nimodipine and omega-conotoxin GVIA blocked fractions of the current and also reduced the magnitude of the responses to DHPG, indicating that both L- and N-type Ca2+ channels were regulated. These results further differentiate the slow modulatory pathway observed in neocortical neurons when Ca2+ is used as the charge carrier from the rapid voltage-dependent mechanism reported to inhibit Ba2+ currents under Ca2+-free conditions.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane- 1,3-dicarboxylic acid [(IS,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-alpha-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and I mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-(alpha-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methylD-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 microM) only the 50 microM infusion significantly decreased dialysate 5-HT. The GABA(A) receptor agonist, bicuculline (30 microM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG's group II/group II mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.     

Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.     

Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors

Cultured dorsal root ganglion neurons were voltage clamped at -90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (betaNAD+), intracellular flash photolysis of caged 3',5'-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of betaNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 microM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 microM glutamate in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 microM dizocilpine maleate (MK 801), or by extracellular application of 10 microM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of betaNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of betaNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves betaNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.     

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.     

Cannabinoid receptor agonists protect cultured rat hippocampal neurons from excitotoxicity

Cannabinoid receptor agonists act presynaptically to inhibit the release of glutamate. Because other drugs with this action are known to reduce excitotoxicity, we tested several cannabimimetics in a model of synaptically mediated neuronal death. Reduction of the extracellular Mg2+ concentration to 0.1 mM evoked a repetitive pattern of intracellular Ca2+ concentration ([Ca2+]i) spiking that, when maintained for 24 hr, resulted in significant neuronal death. The [Ca2+]i spiking and cell death in this model result from excessive activation of N-methyl-D-aspartate receptors, as indicated by the inhibition of both [Ca2+]i spiking and neuronal death by the N-methyl-D-aspartate receptor antagonist CGS19755 (10 microM). The cannabimimetic drug Win55212-2 (100 nM) completely blocked [Ca2+]i spiking and prevented neuronal death induced by low extracellular Mg2+ concentrations. These effects on [Ca2+]i spiking and viability were stereoselective and were prevented by the CB1 receptor antagonist SR141716 (100 nM). The partial agonist CP55940 (100 nM) also afforded significant protection from excitotoxicity. Cannabimimetic drugs did not protect cells from the direct application of glutamate (30 microM). These data suggest that cannabimimetic drugs may slow the progression of neurodegenerative diseases.     

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.     

Novel diversity in Th1, Th2 type differentiation of hemagglutinin-specific T cell clones elicited by natural influenza virus infection in three major haplotypes (H-2b,d,k)

The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.     

NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release

Before action potential-evoked Ca2+ transients, basal presynaptic Ca2+ concentration may profoundly affect the amplitude of subsequent neurotransmitter release. Reticulospinal axons of the lamprey spinal cord receive glutamatergic synaptic input. We have investigated the effect of this input on presynaptic Ca2+ concentrations and evoked release of neurotransmitter. Paired recordings were made between reticulospinal axons and the neurons that make axo-axonic synapses onto those axons. Both excitatory and inhibitory paired-cell responses were recorded in the axons. Excitatory synaptic inputs were blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5; 50 microM). Application of NMDA evoked an increase in presynaptic Ca2+ in reticulospinal axons. Extracellular stimulation evoked Ca2+ transients in axons when applied either directly over the axon or lateral to the axons. Transients evoked by the two types of stimulation differed in magnitude and sensitivity to AP-5. Simultaneous microelectrode recordings from the axons during Ca2+ imaging revealed that stimulation of synaptic inputs directed to the axons evoked Ca2+ entry. By the use of paired-cell recordings between reticulospinal axons and their postsynaptic targets, NMDA receptor activation was shown to enhance evoked release of transmitter from the axons that received axoaxonic inputs. When the synaptic input to the axon was stimulated before eliciting an action potential in the axon, transmitter release from the axon was enhanced. We conclude that NMDA receptor-mediated input to reticulospinal axons increases basal Ca2+ within the axons and that this Ca2+ is sufficient to enhance release from the axons.     

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction. J. Neurophysiol. 80: 2893-2899, 1998. N-Methyl--aspartate (NMDA) reduces release from crayfish excitatory nerve terminals. We show here that polyclonal and monoclonal antibodies raised against the mammalian postsynaptic NMDA receptor subunit 1 stain specifically the presynaptic membrane of release boutons of the crayfish neuromuscular junction. In crayfish ganglionic membranes, the polyclonal antibody recognizes a single protein band that is somewhat larger (by approximately 30 kD) than the molecular weight of the rat receptor. Moreover, the monoclonal (but not the polyclonal) antibody abolishes the physiological effect of NMDA on glutamate release. The monoclonal antibody did not prevent the presynaptic effects of glutamate, which also reduces release by activation of quisqualate presynaptic receptors. Only when 6-cyano-7-nitroquinoxatine-2,3,dione (CNQX) was added together with the monoclonal antibody was the presynaptic effect of glutamate blocked. These results show that presynaptic glutamate receptors of the crayfish NMDA type are involved in the regulation of neurotransmitter release in crayfish axon terminals. Although the crayfish receptor differs in its properties from the mammalian NMDA receptor, the two receptors retained some structural similarity.     

Assessment of the role of glutathione conjugation in the protection afforded by anethol dithiolthione against hexachloro-1,3-butadiene-induced nephrotoxicity

Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca(2+)-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid-dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin- and KCl-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+, it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCl- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C.