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The intraventricular administrations of L-glutamate or trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), which is an agonist for the metabotropic glutamate receptor, induced conformational changes of the smooth endoplasmic reticulum (SER) to form lamellar bodies, consisting of stacks of flattened cisterns in Purkinje cell dendrites of the rat cerebellum. The formation of lamellar bodies by t-ACPD or by anoxia was blocked by pretreatment of L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is an antagonist for the metabotropic glutamate receptor. Injections of N-methyl-D-aspartic acid (NMDA) and amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate, which are categorized as acting on ionotropic receptors of glutamate, did not cause the formation of lamellar bodies, although kainate condensed the dendritic cytoplasm and produced a swelling of surrounding astrocytes. The cisterns of lamellar bodies formed by t-ACPD were long and formed regular stacks. Many intercisternal bridges were arranged with a center-to-center distance of about 25 nm between apposed cisterns. The bridges appeared as short tubes about 15 nm in diameter and in length, a clear center of which linked the lumen of their cisterns. The present results revealed that an excess release of excitatory transmitter by brief anoxia activates metabotropic glutamate receptors, which transform the networks of SER that normally release Ca2+ widely to the neuronal cytoplasm into lamellar bodies. Large Ca2+ storage pools of lamellar bodies are formed by the association of opposing molecules that belong to different cisterns and may protect excess release of Ca2+ from their reservoirs.

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

The anticonvulsant or proconvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in a chemoconvulsant model using pentylenetetrazole (PTZ). Mice received mGluR ligands by intracerebroventricular (i.c.v.) infusion prior to a subcutaneous injection of PTZ and the latency to onset of tonic convulsions was recorded. The group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA) dose-dependently antagonised PTZ-induced seizures with a mean ED50 value of 465 nmol. In contrast, the selective group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine [(S)-DHPG], was proconvulsive and decreased the PTZ-induced seizure latency (ED50=60 nmol i.c.v.). A selective agonist of group II mGluRs, (1S,3S)-1-aminocyclopentane dicarboxylic acid [(1S,3S)-ACPD], was proconvulsive but did not affect PTZ-induced seizure latency. Moreover, the proconvulsant effect of (IS,3S)-ACPD was not blocked by the mGluR2 antagonist, alpha-methylserine-O-phosphate monophenyl ester but was blocked by AIDA suggesting the involvement of group I mGluRs. (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV), which is a potent mGluR2 antagonist and a group III mGluR agonist at higher doses, increased the PTZ-induced seizure latency (ED50=51 nmol) and this effect was fully reversed by the group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4). Similarly, the group III mGluR agonist 1-amino-3-(phosphonomethylene)cyclobutanecarboxylate (cyclobutylene-AP5) increased the PTZ-induced seizure latency (ED50=12 nmol) in a MAP4-sensitive manner. Collectively, these data suggest that mGluR ligands modulate PTZ-induced seizure activity in mice by either antagonizing group I mGluRs or activating group III mGluRs.

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 (±)-α-methyl-4-carboxy-phenylglycine (MCPG), and group III agonistl-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 byl-AP4 (100–200 μm). 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 thesignal-to-noise ratioof oral-related synaptic input to trigeminal motoneurons during rhythmical jaw movements.

Perfusion of hippocampal slices with an inhibitor of nitric oxide (NO) synthase-blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

Metabotropic glutamate receptors (mGluRs) participate in glutamate neural transmission, but their role in the pathophysiology of spinal cord injury (SCI) has not been explored. Accordingly, we examined the role of group I mGluRs, which are linked to phospholipase C, in mediating SCI using an in vitro model. A dorsal column segment was isolated from the spinal cord of adult rats, maintained in vitro, and injured by compression for 15 sec with a clip having a 2 g closing force. Under control conditions after SCI, the compound action potential (CAP) amplitude was reduced to 69.1 +/- 5.4% of baseline. Blockade of group I mGluR receptors with MCPG, 4CPG, or AIDA resulted in improved recovery of CAP amplitude (82.2 +/- 2.0%, 86.2 +/- 3.9%, and 86.0 +/- 2.5% of baseline, respectively). The group I/II agonist trans-ACPD and selective group I agonist DHPG exacerbated the posttraumatic reduction of CAP amplitude. The phospholipase C inhibitor U-73122 improved recovery of CAP amplitude after traumatic spinal cord axonal injury. Western blotting and immunocytochemistry demonstrated the presence of mGluR1alpha-immunopositive astrocytes and the absence of mGluR5 in spinal cord white matter. These studies are consistent with the hypothesis that activation of group I mGluR receptors after SCI exacerbates posttraumatic axonal injury through a phospholipase C dependent mechanism. The presence of mGluR1alpha labeling on astrocytes suggests a role for these cells in the pathophysiology of SCI. Additional studies in vivo, are required to further clarify the role of mGluRs in acute traumatic SCI.

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist ˛ 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(3)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69^78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity. z 1998 Elsevier Science B.V. All rights reserved.

1. Whole-cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). 2. Both high voltage-activated (HVA) and low voltage-activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. 3. The transient LVA calcium current activated rapidly on depolarization (half-activation, -59 mV) and inactivated during maintained depolarization (half-inactivation, -89 mV). This T-type current was observed in somatic recordings but was absent from presynaptic terminals. 4. On the basis of their pharmacological sensitivity, P/Q-type Ca2+ channels accounted for only 6 % of the somatic HVA, while L-, N- and R-type Ca2+ channels each accounted for around one-third of the somatic calcium current. 5. The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. 6. We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P-type contribute little to somatic currents but predominate at the synaptic terminal; L-, N- and R-types dominate at the soma, but contribute negligibly to calcium currents in the terminal.

The mGluR agonist 1S,3R-ACPD induces a potent excitatory inward current in various central neurons, but the underlying mechanism is not fully understood. Thus, we explored the signal transduction mechanism underlying the 1S,3R-ACPD-induced inward current in mouse cerebellar Purkinje cells. Iontophoretic application of 1S,3R-ACPD produced a group I mGluR antagonist-sensitive inward current. This current closely resembled a slow synaptic inward current evoked by repetitive stimulation of parallel fibers. Although phosphoinositide hydrolysis is shown to be coupled with group I mGluRs, we found that intracellular injections of various PLC inhibitors and an IP3 receptor antagonist heparin only partially inhibited the 1S, 3R-ACPD-induced current. Moreover, intracellular injection of the Ca2+ chelator BAPTA or a selective Na+/Ca2+ exchange inhibitor KB-R7943 affected slightly the inward current. In contrast, infusion of GDPbetaS and GTPgammaS markedly suppressed the 1S,3R-ACPD-induced current. These results suggest that activation of mGluR1 in mouse cerebellar Purkinje cells by 1S,3R-ACPD application or by repetitive stimulation of parallel fibres induces an inward current with a minor contribution from intracellular Ca2+ or Na+/Ca2+ exchange.

Na+-K+-2Cl−cotransporters are important in renal salt reabsorption and in salt secretion by epithelia. They are also essential in maintenance and regulation of ion gradients and cell volume in both epithelial and nonepithelial cells. Expression of Na+-K+-2Cl−cotransporters in brain tissues is high; however, little is known about their function and regulation in neurons. In this study, we examined regulation of the Na+-K+-2Cl−cotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitive K+influx, and protein expression was evaluated by Western blot analysis. Glutamate was found to induce a dose- and time-dependent stimulation of Na+-K+-2Cl−cotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonist N-methyl-d-aspartic acid (NMDA) and the metabotropic receptor agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD) significantly stimulated the cotransport activity in these cells. NMDA-mediated stimulation of the Na+-K+-2Cl−cotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate. trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)-α-methyl-(4-carboxyphenyl)glycine. The results demonstrate that Na+-K+-2Cl−cotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

The genes encoding the ionotropic N-methyl- D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMB01 and pMB02. The protease-deficient yeast Saccharomyces cerevisiae cl3-ABYS-86 (leu−, ura−, his−) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes. Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains. By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1−3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88 ± 5.38 nM (NR1a), 1365.11 ± 76 nM (NR1a/NR2D), 22.97 ± 3.37 nM for NR1a/NR2B/NR2D and 7.4 ± 1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07 ± 0.92 (NR1a), 14.63 ± 0.50 (NR1a/NR2D), 12.85 ± 1.68 (NR1a/NR2B/NR2D) and 8.3 ± 0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536 ± 43, 688 ± 60, and 856 ± 48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.

Placental uptake and transport of three nonmetabolizable amino acids with different reactivities for transport systems were studied in sheep under normal physiologic conditions. Methylaminoisobutyric acid (MeAIB), which has specific affinity for the sodium-dependent A system transporters, demonstrated placental concentrative uptake from the uterine and the umbilical circulations, but virtually no transport from mother to fetus. By contrast, aminoisobutyric acid (AIB) and aminocyclopentane-1-carboxylic acid (ACP), which have affinity for both sodium-dependent and sodium-independent transporters, demonstrated both concentrative uptake and transport from mother to fetus. ACP transport rate to the fetus was approximately twice the AIB transport rate. It is concluded that a neutral amino acid which interacts almost exclusively with the weakly reversible system A transporters may be transported rapidly into the placenta and may attain high concentrations within this organ but cannot escape from placenta to fetus down its own concentration gradient because the exit route is controlled by reversible amino acid transporters at the fetal surface of the placenta. Conversely, high affinity for reversible Na-independent transporters may be a necessary condition for the rapid transport of an amino acid from placenta to fetus.

Sustained (20 h) exposure to the glycine partial agonist 1-aminocyclopropanecarboxylic acid (ACPC) significantly reduced N-methyl- d -aspartate (NMDA)-induced neurotoxicity in cultured spinal cord neurons when the NMDA (25 and 100 μM) was added to the cultures 30 min after removal of the ACPC (1 mM). In contrast, ACPC preexposure failed to protect against kainate-induced neuronal injury. The magnitude of neuronal protection against NMDA (100 μM) was further enhanced if the neurons pretreated with ACPC were reexposed to this drug during the NMDA challenge. In addition, the potencies of both the competitive NMDA antagonist AP5 and the noncompetitive antagonist dizocilpine to block NMDA toxicity were significantly increased following ACPC preexposure, while the potency of the competitive glycine receptor antagonist 7-chlorokynurenate (7-CK) was unchanged. Analysis of Northern blots suggest that ACPC-induced changes in NMDA receptor function were not associated with alterations in the levels of the mRNAs encoding the NMDAR-1, -2A, -2B, or -2C subunits. These results indicate that sustained exposure to ACPC modifies NMDA receptors in a manner that diminishes NMDA receptor-mediated neurotoxicity while selectively enhancing the potencies of several NMDA receptor antagonists. These effects do not appear to be related to changes in expression of specific NMDA receptor subunits, and may instead involve a post-translational modification of one or more subunit proteins.

The effects of the selective GABA(B) receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]aminolpropyl] (diethoxymethyl) phosphinic acid (CGP 52432) on muscarinic (mAChR) and metabotropic glutamate (mGluR) responsiveness were studied in slices of piriform cortex from both immature (P16-P22) and adult (or =P40) rats, using a conventional intracellular recording technique. In both adult and immature slices, CGP 52432 (1 microM) had no effect on neuronal membrane properties, whereas it selectively abolished the late inhibitory postsynaptic potential (IPSP) evoked by local electrical stimulation of association fibre terminals. Age-related changes in mAChR (but not mGluR) responsiveness were also detected. In adult neurones, bath-application of the mAChR agonist oxotremorine-M (OXO-M; 10 microM), or the selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 10 microM) evoked similar membrane depolarization and inhibition of evoked excitatory postsynaptic potentials (EPSPs). However, while 1S,3R-ACPD and OXO-M produced indistinguishable slow excitatory effects in immature slices, during superfusion with OXO-M, neurones exhibited spontaneous paroxysmal depolarizing shifts (PDSs) that were suppressed in the presence of atropine (1 microM) or the selective GABA(B) receptor agonist beta-parachlorophenyl-gamma-aminobutyric acid [(-)baclofen; 10 microM]. Also, application of OXO-M resulted in a pronounced prolongation (rather than a decrease) of electrically evoked postsynaptic potentials (PSPs) which now exhibited recurrent superimposed spike discharges. In adult slices, in the continuous presence of CGP 52432 (1 microM; 20 min pre-incubation), a subsequent exposure to 10 microM OXO-M or 1S,3R-ACPD failed to induce any spontaneous epileptiform activity, and evoked PSPs were consistently suppressed. In contrast, in immature slices, after incubation in CGP 52432 (1 microM; 20 min), a subsequent application of a low dose of OXO-M (2.5 microM), which was inactive per se, was able to produce spontaneous PDSs and a prolongation of evoked PSPs. We conclude that a reduction in GABA(B)-mediated synaptic inhibition in immature slices (in co-operation with other factors) may contribute to the facilitation of excitatory neurotransmission and therefore play a role in the generation of mAChR-induced epileptiform activity.

Glutamate suppressed high-voltage-activated barium currents (IBa,HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an ω-conotoxin GVIA-sensitive current. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (l-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized. Many neurotransmitters suppress voltage-activated calcium channels, particularly the high-voltage-activated L- and N-types (Hille, 1994). One function of this inhibition is to autoregulate transmitter release by presynaptic negative feedback. A conventional model is that transmitter release from the presynaptic terminal results in activation of postsynaptic ionotropic receptors and concomitant stimulation of presynaptic metabotropic receptors. However, there are many examples of postsynaptic metabotropic receptors. In fact, the first description of a metabotropic glutamate receptor was the retinal ‘on’ bipolar postsynaptic receptor (Slaughter & Miller, 1981; Nawy & Jahr, 1990; Shiells & Falk, 1990). There is also evidence that ionotropic glutamate receptors may suppress voltage-gated calcium channels (Nistri & Cherubini, 1991). Clearly, narrowly defined roles do not adequately detail the physiology of ionotropic and metabotropic glutamate receptors. We chose to examine the glutamatergic regulation of calcium currents in amphibian retinal ganglion cells. These neurons possess a plethora of glutamate receptors, including the AMPA/kainate and NMDA ionotropic receptors (Slaughter & Miller, 1983) and at least one form of metabotropic receptor (Akopian & Witkovsky, 1996). Additionally, these neurons are thought to be purely postsynaptic in the retina and therefore ionotropic and metabotropic receptors may function in a unique manner to control postsynaptic integration rather than regulation of transmitter release. Recordings from ganglion cells revealed sustained, high-voltage-activated calcium channel currents. These currents were suppressed by both ionotropic and metabotropic glutamate agonists. Ganglion cells possessed all three groups of metabotropic receptor, although group III agonists produced the largest effect on the calcium channel. The metabotropic receptors stimulated calcium release from internal stores, acting through both inositol trisphosphate (IP3) and ryanodine receptors, leading to suppression of L-type calcium channels. The kainate-mediated inhibition of N-type calcium channels probably involves two mechanisms, one mediated by calcium-calmodulin and one by phosphatases.

Glutamate receptors underlying synaptic excitation in the rat's lateral superior olive were studied by whole-cell patch clamp recordings in a brain slice preparation. Recordings from two morphological types of cells, bipolar and multipolar, identified by intracellular labeling with biocytin, showed that there were no obvious differences in responses mediated or modulated by ionotropic and metabotropic receptors between these two types of neurons. The excitatory postsynaptic potentials (EPSPs) elicited by ipsilateral stimulation of the trapezoid body consisted of two components. An earlier component, which had faster rise time constant and decay time constant, was mediated by non-NMDA receptors. A later component, which had slower rise time and decay time constants, was mediated by NMDA receptors. Suprathreshold responses (action potentials), which arose from the early component, were always abolished by the non-NMDA antagonist, CNQX, but not by the NMDA antagonist, APV. These results suggest that both non-NMDA and NMDA receptors are present in LSO neurons, and that fast excitatory transmission in LSO is primarily mediated by non-NMDA receptors. The metabotropic glutamate receptor agonists, t-ACPD and L-AP4, reduced the size of EPSPs evoked by stimulation of the ipsilateral trapezoid body in LSO neurons; the reductive action of t-ACPD was reversed by the antagonist, MCPG, indicating that metabotropic glutamate receptors, probably group II and III subtypes, can modulate excitatory synaptic transmission in LSO.

Chu, Zhiguo and John J. Hablitz. Activation of group I mGluRs increases spontaneous IPSC frequency in rat frontal cortex. J. Neurophysiol. 80: 621–627, 1998. The effect of metabotropic glutamate receptor (mGluR) activation on inhibitory synaptic transmission was examined by using whole cell patch-clamp recordings. Spontaneous (s) and miniature (m) inhibitory postsynaptic currents (IPSCs) were recorded from visually identified layer II/III pyramidal neurons in rat neocortex in vitro. Excitatory postsynaptic currents (EPSCs) were blocked by using bath application of 20 μM d(−)2-amino-5-phosphonovaleric acid and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione. In the presence of 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylic acid (30–100 μM), l-quisqualate (5 μM), and the group I selective mGluR agonist ( S)-3,5-dihydroxyphenylglycine (100 μM), the frequency of sIPSCs was increased. Decay kinetics of sIPSCs were unaffected. No enhancement of mIPSCs was observed. Bath application of group II (2 S,3 S,4 S-α-carboxycyclopropyl-glycine; 5 μM) and group III selective mGluR agonists (l-2-amino-4-phosphonobutyric acid; 100 μM) had no detectable effects on the frequency or amplitude of sIPSCs. These findings indicate that activation of group I mGluRs (mGluR1 and/or mGluR5) enhances γ-aminobutyric acid–mediated synaptic inhibition in layer II/III pyramidal neurons in neocortex. The lack of effect on mIPSCs suggests a presynaptic action via excitation of inhibitory interneurons.

Golshani, Peyman, Richard A. Warren, and Edward G. Jones. Progression of change in NMDA, non-NMDA, and metabotropic glutamate receptor function at the developing corticothalamic synapse. J Neurophysiol. 80: 143–154, 1998. The development of receptor function at corticothalamic synapses during the first 20 days of postnatal development is described. Whole cell excitatory postsynaptic currents (EPSCs) were evoked in relay neurons of the ventral posterior nucleus (VP) by stimulation of corticothalamic fibers in in vitro slices of mouse brain from postnatal day 1 (P1). During P1–P12, excitatory postsynaptic conductances showed strong voltage dependence at peak current and at 100 ms after the stimulus and were almost completely antagonized by dl−2-amino-5-phosphonopentoic acid (APV), indicating that N-methyl-d-aspartate (NMDA) receptor-mediated currents dominate corticothalamic EPSCs at this time. After P12, in 42% of cells, excitatory postsynaptic conductances showed no voltage-dependence at peak current but still showed voltage-dependence 100-ms poststimulus. This voltage-dependent conductance was antagonized by APV. The nonvoltage-dependent component was APV resistant, showed fast decay, and was antagonized by the nonNMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In the remaining 58% of cells after P12, excitatory postsynaptic conductances showed moderate voltage dependence at peak conductance and strong voltage dependence 100 ms after the stimulus. Analysis of EPSCs before and after APV showed a significant increase in the relative contribution of the non-NMDA conductance after the second postnatal week. From P1 to P16, there was a significant decrease in the time constant of decay of the NMDA EPSC but no change in the voltage dependence of the NMDA response. After P8, slow EPSPs, 1.5–30 s in duration and mediated by metabotropic glutamate receptors (mGluRs), could be evoked by high-frequency stimulation of corticothalamic fibers in the presence of APV and CNQX. Similar slow depolarizations could be evoked by local application of the mGluR agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( t-ACPD) but from P0. Both conductances were blocked by the mGluR antagonist, (RS)-α-methyl-4-carboxyphenylglycine. Hence functional mGluR receptors are present on VP cells from birth, but their synaptic activation at corticothalamic synapses can only be detected after P8. In voltage clamp, the extrapolated reversal potential of the t-ACPD current, with potassium gluconate-based internal solution, was +12 ± 10 (SE) mV, and the measured reversal potential with cesium gluconate-based internal solution was 1.5 ± 9.9 mV, suggesting that the mGluR-mediated depolarization was mediated by a nonselective cation current. Replacement of NaCl in the external solution caused the reversal potential of the current to shift to −18 ± 2 mV, indicating that Na+ is a charge carrier in the current. The current amplitude was not reduced by application of Cs+, Ba2+, and Cd2+, indicating that the t-ACPD current was distinct from the hyperpolarization-activated cation current ( I H) and distinct from certain other previously characterized mGluR-activated, nonselective cation conductances.

The relay of information through thalamus to cortex is dynamically gated, as illustrated by the retinogeniculocortical pathway. Important to this is the inhibitory interneuron in the lateral geniculate nucleus (LGN). For the typical neuron, synaptic information arrives through postsynaptic dendrites and is transmitted by axon terminals. However, the typical thalamic interneuron, in addition to conventional axonal outputs, has distal dendrites that serve both pre- and postsynaptic roles. These dendritic terminals participate in curious and enigmatic triadic arrangements, in which each contacts a relay cell dendrite and is contacted by a glutamatergic retinal terminal that innervates the same relay cell dendrite. Here we show that agonists of the metabotropic glutamate receptor (mGluR) activate dendritic terminals of interneurons in the absence of action potentials, thereby inhibiting the postsynaptic relay neuron. Somatic recordings from LGN interneurons reveal that there is no response to mGluR agonists, suggesting that their dendritic terminals are electrically isolated from their somata and axons, consistent with anatomical modelling of these cells. Our results offer insight into the functioning of triadic circuitry and indicate that thalamic interneurons can perform independent computations expressed through axonal as opposed to dendritic outputs.

Stimulation of metabotropic glutamate receptors in vitro has been shown to accelerate the breakdown of amyloid precursor protein (APP) to form increased production of non-amyloidogenic secreted APP (sAPP). The mechanism whereby this occurs is not entirely clear but it is presumed to be linked to generation of diacylglycerol and activation of protein kinase C because other neurotransmitter receptors such as m1 and m3 muscarinic receptors, known to be coupled to this second messenger cascade, likewise increase sAPP production. Although it is presumed that a reciprocal relationship exists between the formation of amyloid beta protein (Abeta) and the production of sAPP, recent evidence suggests alternative processing can occur. Given the fact that much of the observations on APP metabolism have been made in vitro we sought to investigate the effect of metabotropic receptor activation on Abeta in vivo in a species known to contain the same amino acid sequence of Abeta as found in humans. Intrahippocampal injection of the mGluR agonist 1S,3R-ACPD in guinea pigs produced neurodegeneration of CA1 hippocampal pyramidal neurons at 12 h postinjection. Immunocytochemistry of sections from ACPD injected animals using selective antibodies to Abeta revealed the presence of punctate intraneuronal granules in pyramidal neurons of the hippocampus. These structures appeared to be localized within the nucleus and were particularly prominent in neurons within the region of neurodegeneration. Immunoreactivity was not observed in vehicle injected controls nor in sections from ACPD injected animals stained with preadsorbed antiserum. Abeta immunodetection was correlated with the onset of neurodegeneration since animals evaluated at 1 h and 4 h postinjection lacked both Abeta immunoreactivity as well as neurodegeneration. Evaluation of animals injected with NMDA revealed neurodegeneration but no Abeta immunoreactivity suggesting Abeta formation did not appear to be due to non-selective excitotoxicity. Staining of sections with antibodies directed to various regions of APP demonstrated increased C-terminal APP immunoreactivity in pyramidal neurons in the vicinity of degeneration. These data support recent in vitro studies illustrating that Abeta can be found intracellularly within neurons.

BAY 10-8888, a cyclic β-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related β-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans . However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans . To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAAreceptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.

Dopamine neurons in the substantia nigra heavily innervate the striatum, making it the nucleus with the highest levels of dopamine in the adult brain. The present study analyzes the time course and the density of striatal innervation by nigral dopamine neurons and characterizes the role of the neurotransmitter glutamate during the development of the nigrostriatal pathway. For this purpose, organotypic cultures containing the cortex, the striatum, and the substantia nigra (triple cultures) were prepared from rat brains at postnatal day (PND) 0–2 and were cultured for up to 60 din vitro(DIV). Dopamine fibers and neurons were labeled using tyrosine hydroxylase (TH) immunohistochemistry. Striatal TH-ir fiber density was quantitatively analyzed using confocal laser scanning microscopy (CLSM). In long-term triple cultures (44 ± 3 DIV), the striatal dopamine fiber density was high and was weakly correlated with the number of nigral dopamine neurons. The high striatal dopamine fiber density mainly resulted from an enhanced ingrowth and ramification of dopamine fibers from nigral neurons during 8–17 DIV. The metabotropic glutamate receptor (mGluR) antagonistl(+)-2-amino-3-phosphonopropionic acid (l-AP-3) selectively inhibited this dopaminergic innervation of the striatum, whereas ionotropic GluR antagonists had no effect. Thel-AP-3-mediated inhibition was prevented by the mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). The inhibition of the striatal dopaminergic innervation byl-AP-3 was further confirmed by anterograde tracing of the nigrostriatal projection withPhaseolus vulgarisleucoagglutinin. These results indicate that glutamate, by acting on group I mGluRs, plays an important “trophic” role for the development of the nigrostriatal dopamine pathway.

In rat brainstem slices, we investigated the possible role of metabotropic glutamate receptors in modulating the synaptic transmission within the medial vestibular nuclei, under basal and plasticity inducing conditions. We analysed the effect of the metabotropic glutamate receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine on the amplitude of the field potentials and latency of unitary potentials evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation, and on the induction and maintenance of long-term potentiation, after high-frequency stimulation. Two effects were observed, consisting of a slight increase of the field potentials and reduction of unit latency during the drug infusion, and a further long-lasting development of these modifications after the drug wash-out. The long-term effect depended on N-methyl-D-aspartate receptor activation, as D,L-2-amino-5-phosphonopentanoic acid prevented its development. We suggest that (R,S)-alpha-methyl-4carboxyphenylglycine enhances the vestibular responses and induces N-methyl-D-aspartate-dependent long-term potentiation by increasing glutamate release, through the block of presynaptic metabotropic glutamate receptors which actively inhibit it. The block of these receptors was indirectly supported by the fact that the agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid reduced the vestibular responses and blocked the induction of long-term potentiation by high-frequency stimulation. The simultaneous block of metabotropic glutamate receptors facilitating synaptic plasticity, impedes the full expression of the long-term effect throughout the (R,S)-alpha-methyl-4-carboxyphenylglycine infusion. The involvement of such a facilitatory mechanism in the potentiation is supported by its reversible reduction following a second (R,S)-alpha-methyl-4-carboxyphenylglycine infusion. The drug also reduced the expression of potentiation induced by high-frequency stimulation. Conversely the electrical long-term potentiation was still induced, but it was occluded by the previous drug potentiation. We conclude that metabotropic glutamate receptors play a dual functional role in the medial vestibular nuclei, consisting in the inhibition of glutamate release under basal conditions, and the facilitation of N-methyl-D-aspartate-dependent plasticity phenomena.

Kindling induced by 13 intraperitoneal injections of 40 mg/kg pentylenetetrazole (PTZ) over a period of 4 weeks resulted in a significant long-lasting increase in both the convulsive susceptibility of animals to the convulsant and the density of the specific [3H]-L-glutamate binding sites in the hippocampus. The quisqualate- and kainate-sensitive [3H]-L-glutamate binding sites were increased 24 h after the final PTZ injection, whereas the N-methyl-D-aspartate (NMDA)-sensitive sites had only a tendency to be enhanced. Furthermore, we investigated [3H]-L-glutamate binding on metabotropic receptors and found a significant increase in the hippocampus following PTZ kindling. In addition, in hippocampal tissue of kindled rats (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated inositol phosphate formation is increased. It can be concluded that the increase in metabotropic glutamate receptor (mGluR) density may be the expression of a specific enhancement in susceptibility of the glutamatergic systems to this excitatory amino acid developing in the course of PTZ-induced kindling.

The changes in motility, chemotactic responsiveness, and flagellation of Rhizobium meliloti RMB7201, L5-30, and JJ1c10 were analyzed after transfer of the bacteria to buffer with no available C, N, or phosphate. Cells of these three strains remained viable for weeks after transfer to starvation buffer (SB) but lost all motility within just 8 to 72 h after transfer to SB. The rates of motility loss differed by severalfold among the strains. Each strain showed a transient, two- to sixfold increase in chemotactic responsiveness toward glutamine within a few hours after transfer to SB, even though motility dropped substantially during the same period. Strains L5-30 and JJ1c10 also showed increased responsiveness to the nonmetabolizable chemoattractant cycloleucine. Cycloleucine partially restored the motility of starving cells when added after transfer and prevented the loss of motility when included in the SB used for initial suspension of the cells. Thus, interactions between chemoattractants and their receptors appear to affect the regulation of motility in response to starvation independently of nutrient or energy source availability. Electron microscopic observations revealed that R. meliloti cells lost flagella and flagellar integrity during starvation, but not as fast, nor to such a great extent, as the cells lost motility. Even after prolonged starvation, when none of the cells were actively motile, about one-third to one-half of the initially flagellated cells retained some flagella. Inactivation of flagellar motors therefore appears to be a rapid and important response of R. meliloti to starvation conditions. Flagellar-motor inactivation was at least partially reversible by addition of either cycloleucine or glucose. During starvation, some cells appeared to retain normal flagellation, normal motor activity, or both for relatively long periods while other cells rapidly lost flagella, motor activity, or both, indicating that starvation-induced regulation of motility may proceed differently in various cell subpopulations.

Presynaptic metabotropic glutamate receptors (mGluRs) modulate the release of transmitter from most central synapses. However, difficulties in recording from presynaptic structures has lead to an incomplete understanding of the mechanisms underlying these fundamental processes. By recording directly from presynaptic reticulospinal axons and postsynaptic motoneurons of the lamprey spinal cord, we have obtained electrophysiological and optical evidence that vertebrate presynaptic metabotropic glutamate receptors modulate neurotransmitter release at this synapse through two distinct mechanisms: (1) mGluR activation in the presynaptic terminal depresses transmitter release by activating a presynaptic K+ current, and (2) mGluR activation enhances transmitter release by amplifying the action potential–evoked presynaptic Ca2+ signal by rapidly releasing Ca2+ from intracellular stores in a Ca2+-dependent manner. Furthermore, this effect is mediated by physiological release of glutamate from the presynaptic terminals. These autoreceptor–mediated processes are likely to generate complex effects on transmitter release evoked by repetitive stimulation.

We investigated the effects of repeated electroconvulsive shock or imipramine treatment on inositol phosphate accumulation and on the reactivity of neurons to metabotropic glutamate (mGlu) receptor agonists in rat hippocampal slices. (1S,3R)-1-carboxycyclopentane-3-acetic acid (1S,3R-ACPD), a nonselective mGlu receptor agonist, caused a concentration-dependent increase in inositol phosphate in slices from the CA1 region of the hippocampus, an effect that was not modified by imipramine or electroconvulsive shock treatment. 1S,3R-ACPD or the selective agonist of the I group of mGlu receptor, (R,S)-3,5-dihydroxyphenylglycine ((R,S)-3,5-DHPG), produced a concentration-dependent increase of the population spike recorded in the CA1 cell layer. This effect of 1S,3R-ACPD was markedly attenuated by both repeated imipramine and electroconvulsive shock treatment, and the action of (R,S)-3,5-DHPG was markedly attenuated by prolonged imipramine treatment (electroconvulsive shock was not tested). Our results indicate that antidepressant treatment may induce a subsensitivity of group I mGlu receptors when assessed by electrophysiological but not biochemical measures.

To better characterize the roles of metabotropic glutamate receptors (mGluRs) in physiological and pathophysiological processes, there is an important need to learn more about the structural features relevant to the design of novel, high-affinity ligands that are family and subtype specific. To date, many of the biological studies that have been conducted in the area of mGluR research have made use of the agonist (1S,3R)-ACPD. This compound has been shown to act as an agonist at both the group I and group II receptors while showing little selectivity among the four subtypes belonging to these two groups. Moreover, (1S,3S)-ACPD, the cis isomer, shows negligible activity at group I receptors and is a good agonist of mGluR2. Since ACPD is itself somewhat flexible, with four distinctive conformations being identified from molecular modeling studies for the trans isomer and five conformations for the cis isomer, we believed that it would be of interest to examine the activity of an ACPD analogue that has been constrained through the introduction of a single carbon atom bridge. Accordingly, we have prepared an aminobicyclo[2.1.1]hexanedicarboxylic acid (ABHxD-I) analogue of ACPD. The synthesis of this compound was accomplished by use of an intramolecular [2 + 2] photocycloaddition reaction, in which four distinct isomers were isolated. Of these four compounds, only a single isomer, ABHxD-I (6a), was found to be a potent agonist of the mGluRs. This compound, which expresses the fully extended glutamate conformation, was found to be more potent than ACPD at all six of the eight mGluR subtypes that were investigated and to be comparable to or more potent than the endogenous ligand, glutamate, for these receptors. Interestingly, despite its fixed conformation, ABHxD-I, like glutamate, shows little subtype selectivity. Through modeling studies of ABHxD-I (6a), ABHD-VI, LY354740, (1S,3R)-ACPD, (1S, 3S)-ACPD, and l-glutamate, we conclude that the aa conformation of l-glutamate is the active conformation for both group I and group II mGluRs. Moreover, the modeling-based comparisons of these ligands suggest that the selectivity exhibited by LY354740 between the group I and group II mGluRs is not a consequence of different conformations of L-glutamate being required for recognition at these mGluRs but rather is related to certain structural elements within certain regions having a very different impact on the group I and group II mGluR activity. The enhanced potency of ABHxD-I relative to trans-ACPD commends it as a useful starting point in the design of subtype selective mGluR ligands.

In the present study we used freely moving rats with a microdialysis probe placed in their parietal cortex to study the effects of local application of agonists and antagonists of metabotropic glutamate (mGlu) receptors on glutamate release. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 0.1-1 mM), a non-selective agonist of metabotropic glutamate (mGlu) receptors, increased glutamate concentration in the dialysate up to 3-fold. A significant increase in glutamate output in cortical dialysates was also obtained with (RS)-3,5-dihydroxyphenylglycine (DHPG; 0.5-1 mM), a group 1-selective mGlu receptor agonist, suggesting the involvement of group 1 mGlu receptors in 1S,3R-ACPD effects. S-4-carboxyphenylglycine (S-4CPG; 0.3 microM), a mGlu1 receptor antagonist with a mild agonist action on mGlu2 receptors, antagonised, in a surmountable manner, the effects of 1S,3 R-ACPD. Similarly, 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.03-1 mM) a selective group 1 antagonist with a preferential action on mGlu1 type receptors, antagonised the effects of 1S,3R-ACPD. Finally, (S)-(+)-2-(3'-Carboxybicyclo[1.1.1]pentyl)-glycine (UPF596; 30-300 microM), a potent mGlu1 antagonist with modest agonist activity on mGlu5, antagonised 1S,3R-ACPD-induced glutamate release. In conclusion, our data showed that 1S,3R-ACPD-induced glutamate release in the parietal cortex is mediated by mGlu1 receptors and that, under basal conditions, these receptors are not tonically activated.

Previous work has shown that responses of thalamic neurons in vivo to the metabotropic glutamate receptor agonists 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine can be reduced by a variety of phenylglycine antagonists. Responses of thalamic neurons to noxious thermal somatosensory stimuli were reduced in parallel by these antagonists, indicating that these responses are mediated by Group I metabotropic glutamate receptors (i.e. metabotropic glutamate receptor-1 and/or metabotropic glutamate receptor-5), which are known to be linked to phosphoinositol phosphate hydrolysis. The recent development of S-2-methyl-4-carboxyphenylglycine as an antagonist which is highly selective for metabotropic glutamate receptor-1 compared to metabotropic glutamate receptor-5 on human receptors expressed in AV-12 cells, now offers the possibility of discriminating between these two receptor subtypes in order to distinguish which is involved in thalamic responses. We have made recordings from single somatosensory neurons in the thalamus of the rat, and find that S-2-methyl-4-carboxy-phenylglycine is able to reduce responses of neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate, S-3,5-dihydroxyphenylglycine, and noxious stimuli without significant effect on responses to either N-methyl-D-aspartate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. These results suggest that excitatory responses of thalamic neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine may be mediated by metabotropic glutamate receptor-1. Furthermore, the reduction of nociceptive responses by S-2-methyl-4-carboxy-phenylglycine indicates that metabotropic glutamate receptor-1 is involved in thalamic nociceptive processing and that such antagonists may have analgesic properties.

The changes in mean arterial pressure (MAP) and heart rate (HR) in response to the activation of metabotropic receptors in the nucleus tractus solitarii (NTS) with trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-(+/-)-ACPD) were evaluated in conscious and anesthetized Wistar, male rats weighing 240-260 g (N = 8). The responses obtained with trans-(+/-)-ACPD were compared with the responses to L-glutamate (1 nmol/100 nl), since in a previous study we showed that anesthesia converted a pressor response to L-glutamate microinjected into the NTS of conscious rats to a depressor response in the same rats under urethane or chloralose anesthesia. Microinjection of 3 doses of trans-(+/-)-ACPD (100, 500 and 1000 pmol/100 nl) produced a dose-dependent fall in MAP (range, -20 to -50 mmHg) and HR (range, -30 to -170 bpm) under both conscious and chloralose anesthesia conditions. These data indicate that the cardiovascular responses to the activation of metabotropic receptors by trans-(+/-)-ACPD are not affected by chloralose anesthesia while the cardiovascular responses to the activation of excitatory amino acid (EAA) receptors by L-glutamate are significantly altered.

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]iin either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR11XX, NR10XX, and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1 S, 3 R-dicorboxylate (1 S,3 R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]iincreases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Breakwell, N. A., M. J. Rowan, and R. Anwyl. (+)-MCPG blocks induction of LTP in CA1 of rat hippocampus via agonist action at an mGluR group II receptor. J. Neurophysiol. 79: 1270–1276, 1998. We investigated the effect of metabotropic glutamate receptor (mGluR) ligands on the induction of long-term potentiation (LTP) of field excitatory postsynaptic potentials (EPSPs) in CA1 of rat hippocampus, in particular the manner by which the nonsubtype selective mGluR ligand α-methyl-4-carboxyphenylglycine [(+)-MCPG] blocks LTP induction. Normalized control LTP was blocked by (+)-MCPG (250 μM), but not by the mGluRI selective antagonist (S)-4-carboxyphenylglycine (4-CPG), the mGluRII selective antagonist 1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine (MCCG), or the mGluRIII antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid/α-methyl (MAP4). In contrast the mGluRII agonist {(1S,3S)-1-aminocyclopentante-1,3-dicarboxylic acid [(1S,3S)-ACPD]; 10 or 25 μM} completely and consistently blocked LTP. The block of LTP by both (1S,3S)-ACPD and (+)-MCPG could be prevented by preincubation with the mGluRII antagonist MCCG. These studies demonstrate that (+)-MCPG blocks LTP induction through an agonist action at an mGluRII receptor and not through a nonselective antagonist action.

The human neuroblastoma cell line SH-SY5Y was used to study the regulation of methionine adenosyltransferase (MAT II; E.C.2.5.1.6.) catalytic activity and transcript levels in cells of neuronal origin. The cells were exposed for 24 hr to a medium containing different concentrations of methionine (MAT substrate) as well as medium deficient of methionine. Furthermore, cells were treated with hydroxycobalamin, SAM, and the competitive MAT inhibitor cycloleucine. The MAT catalytic activity was inversely correlated to methionine concentrations, e.g. MAT Vmax increased 2-fold in cells grown in methionine-deficient medium as compared with cells cultured under standard conditions. Interestingly, MAT Km also increased from 9.04 +/- 0.44 to 12.08 +/- 0.83 in the methionine-deficient medium. Hydroxycobalamin caused an increase in activity at 40 microM while a decrease was observed at higher concentrations (100, 200, and 400 microM). Cycloleucine caused a significant inhibition of MAT catalytic activity, i.e. the inhibition was approximately 50% in the presence of 4 mM cycloleucine. The relevance of these results for the understanding of observations on MAT catalytic activity in brains of patients with Alzheimer's disease is discussed.

Mossy fiber synaptic transmission at hippocampal CA3 pyramidal cells and interneurons was compared in rat brain slices to determine whether mossy terminals are functionally equivalent. Tetanic stimulation of mossy fibers induced long-term potentiation in pyramidal neurons but was either without effect or it induced depression at synapses onto interneurons. Unlike transmission onto pyramidal neurons, transmission onto interneurons was not potentiated after adenosine 3′,5′-monophosphate (cAMP) activation. Furthermore, metabotropic glutamate receptor depression of transmission onto interneurons did not involve cAMP-dependent pathways. Thus, synaptic terminals arising from a common afferent pathway do not function as a single compartment but are specialized, depending on their postsynaptic target.

Amos, Brian J. and Mitchell Chesler. Characterization of an intracellular alkaline shift in rat astrocytes triggered by metabotropic glutamate receptors. J. Neurophysiol. 79: 695–703, 1998. The modulation of intracellular pH by activation of metabotropic glutamate receptors was investigated in cultured and acutely dissociated rat astrocytes. One minute superfusion of 100 μM (1S,3R)-1-aminocyclopentane-1,3-dicarboxcylic acid (ACPD) evoked an alkaline shift of 0.13 ± 0.013 (mean ± SE) and 0.16 ± 0.03 pH units in cultured (cortical or cerebellar) and acutely dissociated cortical astrocytes, respectively. Alkalinizations were elicited by concentrations of ACPD as low as 1 μM. The ACPD response was mimicked by S-3-hydroxyphenylglycine (3-HPG) and by (s)-4-carboxy-3-hydroxyphenylglycine (4C-3HPG) but was not blocked by α-methyl-4-carboxyphenylglycine (MCPG) or (RS)-1-aminoindan-1,5-dicarboxcylic acid (AIDA), features consistent with an mGluR5 receptor–mediated mechanism. The ACPD-evoked alkaline shift was insensitive to amiloride, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and the v-type ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), bafilomycin, and concanamycin. The alkaline response persisted in Na+- or Cl−-free saline, but was reversibly blocked in bicarbonate-free, N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES)-buffered solutions. A bicarbonate-dependent and Na+-independent alkaline shift could also be elicited by either 3 mM caffeine or 1 μM ionomycin. These data suggest that a rise in cytosolic Ca2+activity is instrumental in triggering the alkalinizing mechanism and that this response is independent of the classic depolarization-induced alkalinization mediated by electrogenic sodium-bicarbonate cotransport.

Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were studied in rat cerebellar granule cell cultures. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. Neither the basal level of extracellular glutamate nor the increased accumulation of glutamate stimulated by KCl (40 mM) or by ionotropic glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainate (KA) (100 microM each), in cells prepared from the ethanol-fed group was significantly different from that in cells prepared from the pair-fed group. Glutamate accumulation stimulated by quisqualate (QA, 100 microM) or by trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 250 microM) in the ethanol-fed group was higher than that in the pair-fed group by 116 and 36%, respectively. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM), an ionotropic QA receptor antagonist, the QA-induced accumulation of glutamate in the ethanol-fed group was still higher than that in the pair-fed group. In the presence of MK-801 (5 microM), an antagonist of the NMDA receptor, the enhanced accumulation of glutamate stimulated by either QA or t-ACPD was still observable in the ethanol-fed group as compared to the pair-fed group. Addition of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM), a selective antagonist of the metabotropic glutamate receptor, abolished the enhanced accumulation of glutamate stimulated by either QA or t-ACPD in the ethanol-fed group. Although immunoblotting of mGluR1 and mGluR2/3 did not show apparent differences between the pair-fed and the ethanol-fed groups, the overall results suggest that the effect of prenatal ethanol exposure was selectively through a pathway mediated by the metabotropic glutamate receptor.

Isomeric norbornane-derived rigid analogs mimicking different potential conformations of ACPD (1-aminocyclopentane-1,3-dicarboxylic acid) and glutamic acid have been synthesized, via the hydantoin route, to be used as conformational probes for bioactive conformations at the glutamatergic receptors of the central nervous system. Activities on metabotropic receptors mGluR1 and mGluR2 are reported and discussed.

Increasing [Ca2+]e from 1.3 4 mM had little effect on basal phospholipase C activity in baby hamster kidney (BHK) cells stably expressing either type 1alpha metabotropic glutamate (mGlu1alpha) or M3-muscarinic (m3) receptors, but enhanced agonist-stimulated phosphoinositide hydrolysis in BHK-mGlu1alpha, but not BHK-m3 cells, demonstrating that Ca2+(e) selectively modulates phosphoinositide signalling stimulated by this receptor subtype.

Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i)1,2 which could represent the start of back-signalling to neurons3,4,5. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.

Svirskis, Gytis and Jørn Hounsgaard. Transmitter regulation of plateau properties in turtle motoneurons. J. Neurophysiol. 79: 45–50, 1998. In motoneurons, generation of plateau potentials is promoted by modulators that block potassium channels. In voltage-clamp experiments with triangular voltage ramp commands, we show that cis-(±)-1-aminocyclopentane-1,3-dicarboxylic acid ( cis-ACPD) and muscarine promote the generation of plateau potentials by increasing the dihydropyridine sensitive inward current, by increasing the input resistance, and by depolarizing the resting membrane potential. Type I metabotropic glutamate receptors (mGluR I) mediate the effects of cis-ACPD. Baclofen suppresses generation of plateau potentials by decreasing the dihydropyridine sensitive inward current, by decreasing the input resistance, and by hyperpolarizing the resting membrane potential. These results suggest that membrane properties of motoneurons are continuously modulated by synaptic activity in ways that may have profound effects on synaptic integration and pattern generation.

The role of metabotropic glutamate receptors in the processing of somatosensory information was studied in dorsal horn neurons of the rat spinal cord. Activation of metabotropic glutamate receptors by local iontophoresis of (1 S ,3 R )-1-amino-cyclopentane-1,3-dicarboxylic acid resulted in an increased response of dorsal horn neurons to ionotropic glutamate receptor agonists ( N -methyl- d -aspartate and kainic acid) applied by iontophoresis. Greater amounts of 1 S ,3 R -1-amino-cyclopentane-1,3-dicarboxylic acid, ejected at high iontophoresis currents, directly excited dorsal horn neurons. Application of (1 S ,3 R )-1-amino-cyclopentane-1,3-dicarboxylic acid also led to a significant increase in responses to innocuous (brush, pressure) but not in responses to noxious (pinch, squeeze) mechanical stimulation. The excitatory effects of (1 S ,3 R )-1-amino-cyclopentane-1,3-dicarboxylic acid were selectively blocked by ( S )-4-carboxy-3-hydroxyphenyl-glycine, an antinociceptive phenylglycine derivative which is a selective group I metabotropic glutamate receptor antagonist, confirming the involvement of these receptors. In wide dynamic range neurons, wind-up, the progressive potentiation of C-fibre-evoked responses during a train of stimuli, was increased by iontophoretic application of (1 S ,3 R )-1-amino-cyclopentane-1,3-dicarboxylic acid or decreased by iontophoresis of ( S )-4-carboxy-3-hydroxyphenyl-glycine without significant change in the C-fibre input. The results suggest an interaction between metabotropic and ionotropic glutamate receptors in spinal dorsal horn neurons. Metabotropic glutamate receptors proved to be involved in the frequency-dependent potentiation of C-fibre responses possibly via modulation of ionotropic glutamate receptors. The long-lasting effects of (1 S ,3 R )-1-amino-cyclopentane-1,3-dicarboxylic acid on wind-up and on responses to peripheral mechanical stimuli strongly support the view that metabotropic glutamate receptors in these neurons may play a significant role in spinal synaptic plasticity, and therefore, may contribute to the central sensitization during mechanical hyperalgesia.

In this study, the effects of glutamate and glutamate receptor agonists in cultured chromaffin cells from bovine adrenal medulla were investigated. It was found that glutamate increases basal catecholamine (CA) secretion in a dose-dependent manner. This effect is mimicked by specific agonists of the four known glutamate receptors N-methyl-D-aspartate (NMDA), quisqualate/(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and trans-(+)-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD), which increased both basal and nicotine-evoked CA secretion. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of KA and AMPA receptors, and L-(+)-2-amino-3-phosphonopropionic acid, an antagonist of the t-ACPD receptor, inhibited the stimulatory effect of related glutamate agonists. Hexamethonium, an antagonist of the nicotinic receptor, failed to influence glutamate agonists except for a 15% inhibition of KA. The increase in CA secretion produced by a 100 µM concentration of glutamate agonists was about 20–60% of that obtained with 10 µM of nicotine, an agonist of the physiological stimulatory cholinergic receptor. The increase in CA secretion produced by glutamate was accompanied by both an increase in bisoxonol fluorescence, suggesting membrane depolarization, and by an increase in intracellular Ca2+ concentrations. Results obtained with image analysis on single cells indicated that the percentage of cells which respond to the stimulation of 50 µM of glutamate is 42%. From these results, we conclude that glutamate, through its four known glutamate receptors, can increase both basal and nicotine-evoked CA secretion in chromaffin cells by a process which involves membrane depolarization and an increase in intracellular calcium levels.

The effects of metabotropic glutamate receptor (mGluR) subtype selective compounds on the excitability of sympathetic preganglionic neurones (SPNs) were investigated. Non-selective mGluR agonists (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid and (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, induced dose-dependent depolarisations in 96 and 75% of SPNs, respectively and hyperpolarisations in 2 and 21% of SPNs. Both agonists could induce subthreshold membrane potential oscillations in previously non-oscillating SPNs and either increased or reduced the frequency of spontaneously occurring oscillations. A selective group I mGluR agonist, 3,5-dihydroxyphenylglycine, depolarised all SPNs tested, induced oscillations in membrane potential of otherwise non-oscillating SPNs and increased the frequency of spontaneous oscillations. Agonists with selectivity for group II mGluRs (1S,3S)-aminocyclopentane-1,3-dicarboxylic acid and (S)-4-carboxy-3-hydroxy-phenylglycine ((S)-4C3HPG) did not induce depolarising responses. However (S)-4C3HPG induced hyperpolarising responses associated with a reduction in the frequency of spontaneous oscillations in two of six SPNs tested. Depolarising and hyperpolarising responses were maintained in the presence of tetrodotoxin indicating a direct action of the agonists upon SPNs. In individual SPNs responses of opposite polarity could be induced from the same initial membrane potential using different agonists, indicating that the opposing responses involved different ionic mechanisms. The broad spectrum mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and the selective group I mGluR antagonist (S)-4-carboxyphenylglycine reversibly depressed mGluR agonist induced depolarisations. These results indicate that SPNs express two mGluR populations with opposing actions on neuronal excitability: group I mGluRs depolarise SPNs and can drive oscillatory membrane potential activity; a minority of SPNs express group II mGluRs which mediate membrane hyperpolarisations and reduce the frequency of membrane potential oscillations.

We have investigated the effect of a number of group I, II and III metabotropic glutamate (mGlu) receptor agonists and antagonists on paired pulse depression in the medial perforant path of the rat dentate gyrus in vitro. A triphasic pattern of a large depression at short intervals (10-50 ms), a reduction of this depression at intermediate intervals (50-200 ms) and again a large depression at late intervals (200 ms) was observed. The group I mGlu receptor agonist, (S)-3,5-dihydroxy phenylglycine ((S)-DHPG; 20 microM) had no significant effect on paired pulse depression at any interstimulus intervals. The mGlu receptor group II and III agonists, L-CCG-1 ((2S,3S,4S)-alpha-(carboxy-cyclopropyl)-glycine), DCG-IV ((2S,1'R,2'R,3'R)-2-2',3'-dicarboxy cyclopropylglycine), 1S,3R-ACPD (1S,3R-1-aminocyclopentate-1,3-dicarboxylic acid) and L-AP4 (L-2-amino-4-phosphono butyric acid) reduced paired pulse depression at interstimulus intervals of 200 ms or less. Application of the non specific mGlu receptor antagonist, MCPG (alpha-methyl carboxy-phenylglycine; 200 microM) completely inhibited the 1S,3R ACPD-induced reduction in paired pulse depression but was without effect on the L-AP4 response. The relatively specific group II antagonist MCCG ((2S,3S,4S)-2-methyl-2-carboxy cycloproprylglycine) at 200 microM and 500 microM, attenuated but did not completely inhibit the DCG-IV induced reduction of paired pulse depression. The putative group III pre-synaptic mGlu receptor antagonist alpha-methyl-L-AP4 and MSOP ((RS)-alpha-methylserine-O-phosphate) both at 200 microM inhibited the L-AP4-induced reduction in paired pulse depression at intermediate phase interstimulus intervals but not at early interstimulus intervals. These results specifically demonstrate the involvement of group III and III mGlu receptor ligands in the modulation of paired pulse depression in the medial perforant pathway.

Martin, Gilles, Zhiguo Nie, and George R. Siggins. Metabotropic glutamate receptors regulate N-methyl-d-aspartate–mediated synaptic transmission in nucleus accumbens. J. Neurophysiol. 78: 3028–3038, 1997. We recorded intracellularly from core nucleus accumbens (NAcc) neurons in brain slices to study the regulation by metabotropic glutamate receptors (mGluRs) of pharmacologically isolated N-methyl-d-aspartate–mediated excitatory postsynaptic currents (NMDA-EPSCs). Monosynaptic NMDA-EPSCs, evoked by local stimulation, were isolated by superfusion of the non-NMDA and γ-aminobutyric acid-A (GABAA) receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and bicuculline (15 μM), respectively. Trans-1-aminocyclopentane-1,3-decarboxylic acid ( trans-ACPD; 50 μM), a nonspecific group 1 and 2 mGluR agonist, had no effect on resting membrane potential (RMP) or input resistance of NAcc neurons. However, it consistently decreased NMDA-EPSC areas (time integrals) dose dependently (1–100 μM; EC50 = 8 μM) and reversibly. The specific group 1 mGluR agonists quisqualate (1–4 μM) and (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 μM) did not mimic the trans-ACPD effect on NMDA-EPSCs, nor did exposure of the slice to the group 1 mGluR antagonist l(+)-2-amino-3-phosphonopropionic acid (l-AP3, 0.4 mM) inhibit the trans-ACPD effect. The putative mGluR1 and mGluR2 antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) at 0.5 mM failed to antagonize trans-ACPD effects but at 1 mM blocked them. Both the group 2 mGluR agonist (2S,3S,4S)-α-(carboxycyclopropyl)-glycine (l-CCG-I, 2 μM) and the group 3 mGluR specific agonist l(+)-2-amino-4-phosphonobutyric acid (l-AP4, 20 μM) attenuated NMDA-EPSC areas; the effect of l-AP4 was blocked by the group 3 antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4; 0.5 mM). Exogenously applied NMDA, in the presence of tetrodotoxin to prevent presynaptic effects, induced inward currents that were decreased by 20 μM l-AP4 but not by 10 μM trans-ACPD. These findings suggest that NMDA receptor-mediated neurotransmission in NAcc is under dual inhibitory regulation by group 2 and 3 metabotropic receptor subtypes: l-AP4–sensitive receptors located postsynaptically and those sensitive to trans-ACPD located presynaptically.

The goal of this study was to evaluate the differences and define the advantages of imaging experimental brain tumors in rats with two nonmetabolized amino acids, 1-aminocyclopentane carboxylic (ACPC) acid and α-aminoisobutyric (AIB) acid compared with imaging with fluorodeoxyglucose (FDG) or the gallium-diethylenetriaminepentaacetic acid chelate (Ga-DTPA). 1-aminocyclopentane carboxylic acid, AIB, and FDG autoradiograms were obtained 60 minutes after intravenous injection to simulate positron emission tomography (PET) imaging, whereas the Ga-DTPA autoradiograms were obtained 5 or 10 minutes after injection to simulate gadolinium (Gd)-DTPA–enhanced magnetic resonance (MR) images. Three experimental tumors were studied (C6, RG2, and Walker 256) to provide a range of tumor types. Triple-label quantitative autoradiography was performed, and parametric images of the apparent distribution volume (Va, mL/g) for ACPC or AIB, relative glucose metabolism (R, μmol/100 g/min), vascular permeability to Ga-DTPA (K1, μL/min/g), and histology were obtained from the same tissue section. The four images were registered in an image array processor, and regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images. A comparative analysis of all measured values was performed. The location and morphologic characteristics of the tumor had an effect on the images and measurements of Va, R, and K1. Meningeal extensions of all three tumors consistently had the highest amino acid uptake (Va) and vascular permeability (K1) values, and subcortical portions of the tumors usually had the lowest values. Va and R (FDG) values generally were higher in tumor regions with high-cell density and lower in regions with low-cell density. Tumor areas identified as “impending” necrosis on morphologic criteria consistently had high R values, but little or no change in Va or K1. Tumor necrosis was seen consistently only in the larger Walker 256 tumors; low values of R and Va for AIB (less for ACPC) were measured in the necrotic-appearing regions, whereas K1 was not different from the mean tumor value. The highest correlations were observed between vascular permeability (K1 for Ga-DTPA) and Va for AIB in all three tumors; little or no correlation between vascular permeability and R was observed. The advantages of ACPC and AIB imaging were most convincingly demonstrated in C6 gliomas and in Walker 256 tumors. 1-aminocyclopentane was substantially better than FDG or Ga-DTPA for identifying tumor infiltration of adjacent brain tissue beyond the macroscopic border of the tumor; ACPC also may be useful for identifying low-grade tumors with an intact blood–brain barrier. Contrast-enhancing regions of the tumors were visualized more clearly with AIB than with FDG or Ga-DTPA; viable and necrotic-appearing tumor regions could be distinguished more readily with AIB than with FDG. [11C]-labeled ACPC and AIB are likely to have similar advantages for imaging human brain tumors with PET.

Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at ≈1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to ≈30% and ≈10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1–5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD andl-AP4 and found them to be reversed in a dose-dependent manner by (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 μm, CPPG completely reversed the effects ofl-AP4 and (1S, 3S)-ACPD, but reduced 5–10 Hz elicited depression by only ≈6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5–10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.

Zhou, Fu-Ming and John J. Hablitz. Metabotropic glutamate receptor enhancement of spontaneous IPSCs in neocortical interneurons. J. Neurophysiol. 78: 2287–2295, 1997. Using neocortical layer I neurons as a model for GABAergic interneurons, we have studied γ-aminobutyric acid-A (GABAA) receptor-mediated spontaneous inhibitory postsynaptic currents (IPSCs) and modulation by metabotropic glutamate receptors (mGluRs). In the presence of 0.5 μM tetrodotoxin (TTX) and ionotropic glutamate receptor antagonists and under symmetrical Cl− conditions, the mean amplitude of miniature IPSCs (mIPSCs) was ∼50 pA at a holding potential of −70 mV with individual events ranging from 10 to 400 pA. Averaged mIPSCs had a 10–90% rise time of ∼0.6 ms. The decay was double exponential. The fast component had a time constant of ∼4 ms and comprised ∼40% of the total amplitude. The slow component had a time constant of ∼22 ms. The frequency of spontaneous IPSCs (sIPSCs), recorded in the absence of TTX, was increased by bath application of the mGluR agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 10–100 μM) or the group I mGluR selective agonist quisqualic acid (Quis; 0.5–1 μM). Under identical conditions, mIPSCs were not affected. The kinetics of sIPSCs and mIPSCs were not altered by ACPD or Quis. Quis (1 μM) induced an inward current of ∼70 pA at a holding potential of −70 mV, whereas ACPD (40–200 μM) induced a smaller inward current. This current was linear over the voltage range −70 to +30 mV and reversed polarity near 0 mV. In current-clamp recordings, both Quis and ACPD induced a depolarization and action potential firing in layer I and deeper layer interneurons. We conclude that neocortical layer I neurons receive GABAA receptor-mediated inhibitory synaptic inputs. Activation of mGluRs, possibly mGluR1 and/or mGluR5, causes an enhancement of inhibitory synaptic transmission by directly depolarizing corticalGABAergic interneurons through the opening of nonselective cation channels.