A metabotropic glutamate receptor regulates transmitter release from cone presynaptic terminals in carp retinal slices.

The role of group III metabotropic glutamate receptors (mGluRs) in photoreceptor-H1 horizontal cell (HC) synaptic transmission was investigated by analyzing the rate of occurrence and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in H1 HCs uncoupled by dopamine in carp retinal slices. Red light steps or the application of 100 microM cobalt reduced the sEPSC rate without affecting their peak amplitude, which is consistent with hyperpolarization or the suppression of Ca(2+) entry into cone synaptic terminals reducing vesicular transmitter release. Conversely, postsynaptic blockade of H1 HC AMPA receptors by 500 nM CNQX reduced the amplitude of sEPSCs without affecting their rate. This analysis of sEPSCs represents a novel methodology for distinguishing between presynaptic and postsynaptic sites of action. The selective agonist for group III mGluRs, l-2-amino-4-phosphonobutyrate (L-APB or L-AP4; 20 microM), reduced the sEPSC rate with a slight reduction in amplitude, which is consistent with a presynaptic action on cone synaptic terminals to reduce transmitter release. During L-APB application, recovery of sEPSC rate occurred with 500 microM (s)-2-methyl-2-amino-4-phosphonobutyrate (MAP4), a selective antagonist of group III mGluR, and with 200 microM 4-aminopyridine (4-AP), a blocker of voltage-dependent potassium channels. Whole-cell recordings from cones in the retinal slice showed no effect of L-APB on voltage-activated Ca(2+) conductance. These results suggest that the activation of group III mGluRs suppresses transmitter release from cone presynaptic terminals via a 4-AP-sensitive pathway. Negative feedback, operating via mGluR autoreceptors, may limit excessive glutamate release from cone synaptic terminals.     

Metabotropic glutamate receptors are required for the induction of long-term potentiation.

Recent observations have led to the suggestion that the metabotropic glutamate receptor may play a role in the induction or maintenance of long-term potentiation (LTP). However, experimental evidence supporting a role for this receptor in the induction of LTP is still inconclusive and controversial. Here we report that, in rat dorsolateral septal nucleus (DLSN) neurons, which have the highest density of metabotropic receptors and show functional responses, the induction of LTP is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate, but is blocked by two putative metabotropic glutamate receptor antagonists, L-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyrate. Furthermore, superfusion of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, a selective metabotropic glutamate agonist, resulted in a long-lasting potentiation of synaptic transmission similar to that induced by tetanic stimuli. Our results demonstrated that activation of postsynaptic metabotropic receptors is both necessary and sufficient for the induction of LTP in the DLSN, and we suggest that such a mechanism may be important at other CNS synapses.     

The role of group II and III metabotropic glutamate receptors in modulation of miniature synaptic activity in frog spinal cord motoneurons

The results of present work demonstrate significant modulating effects mediated by group II and III mGluRs on miniature postsynaptic potentials (mPSP) of the frog spinal motoneurons. The mode of group II and III mGluRs ligands influences, i. e. the changes in the mPSPs average frequency without significant changes in their average amplitude, suggests the presynaptic mechanism of modulation by the change in transmitter release. Selective antagonists of group II and III mGluRs (EGLU and MAP4) increased the average frequency of mPSPs by 52.8 +/- 30.2% (in 4 of 6 motoneurons) and by 54.7 +/- 23.7% (in all 7 motoneurons), respectively. Application of the group III mGluRs agonist LAP4 decreased the mPSPs frequency by 21.8 +/- 5.2% in 3 of 5 motoneurons. The efficiency of the antagonist usage and comparative low efficiency of the agonist suggest that presynaptic mGluRs at motoneuronal synapses under normal condition possess some level of tonic activity. The lack of group II mGluR antagonist effect on some motoneurons appears to be explained by specific localization of the group II mGluRs in preterminal area which is distant from the transmitter release site. The hetero-receptor modulation of pharmacologically isolated inhibitory miniature activity and its glycine- and GABAergic fractions by group III mGluRs was investigated. MAP4 application has been shown to increase the glycine-mediated mlPSPs frequency more than GABA-mediated mlPSPs frequency: in average by 97.6 +/- 20.7% (n = 7) and 54.6 +/- 20.8% (n = 5), respectively. This difference may be due to the segregation of the postsynaptic glycine- and GABA-receptors. The preliminary examination of the convergence of the presynaptic mGluRs and metabotropic GABA(B) receptors influences on GABA-mediated IPSPs was undertaken. It has been shown that presynaptic GABA(B) receptors are tonically active under normal condition. Under condition of GABA(B) receptor blockage by phaclofen, the application of group III mGluR agonist L-AP4 elicited typical effect which was completely taken off by subsequent application of the group III mGluRs antagonist MAP4. This result is in accordance with the assumption that the effects mediated by GABA(B) receptors and mGluRs are independent.     

NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR

Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.     

Role of group-II and -III metabotropic glutamate receptors in the modulation of miniature synaptic activity in frog spinal-cord motoneurons

Results of the present work demonstrate the pronounced modulating effects mediated by group-II and-III metabotropic glutamate receptors (mGluRs) on miniature postsynaptic potentials (mPSPs) of frog spinal motoneurons. The character of the effects of the group-II and-III mGluRs ligands, i.e., changes in the mPSPs frequency and the absence of significant changes in their amplitude, indicates the presynaptic mechanism of the modulation due to a change of the process of transmitter release. The application of ethylglutamate (EGLU) and methylaminophosphobutyrate (MAP4), which are selective antagonists of group-II and-III mGluRs, increased frequency of mPSPs by an average of 52.8 ± 30.2% (in four out of six motoneurons) and by 54.7 ± 23.7% (in all 7 motoneurons), respectively. The application of group-III mGluRs agonist L-aminophosphobutyrate (L-AP4) decreased the mPSP frequency by 21.8 ± 5.2% in three out of five motoneurons. The efficiency of the use of an antagonist and the comparatively low efficiency of the agonist suggest that presynaptic mGluRs are tonically activated during motoneuronal synapses. The absence of a group-II mGluR antagonist effect in some motoneurons appears to be explained by the specific localization of group-II mGluRs in the preterminal area distant from the transmitter release site. The modulation of pharmacologically isolated inhibitory miniature activity and its glycine and GABAergic fractions due to the group-III mGluRs-mediated heteroreceptor was investigated. The MAP4 application was shown to increase the glycine-mediated mIPSPs frequency to a greater degree than the GABA-mediated mIPSPs frequency, as their modulations were equal to an average of 97.6 ± 20.7% (n = 7) and 54.6 ± 20.8% (n = 5), respectively. This difference might possibly be due to the segregation of the postsynaptic glycine and GABA_A receptors. The study of the convergence of the modulating effects of the presynaptic mGluRs and metabotropic GABA_B receptors has shown that, under the condition of the blockage of the tonically active GABA_B receptor by phaclofen, the application of the group-III mGluR agonist L-AP4 produces the typical effect, which was completely eliminated by subsequent application of the group-III mGluRs antagonist MAP4. This result agrees with the point of view regarding the independence of effects mediated by GABA_B receptors and group-III mGluRse.     

Simulation of postsynaptic glutamate receptors reveals critical features of glutamatergic transmission

Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following high-frequency stimulation. They also provide a new tool to analyze the interactions between metabotropic and ionotropic glutamate receptors.     

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.     

Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl) glycine (DCGIV; 5 microM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 microM), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 microM) was combined with a cell-permeant PKA inhibitor H-89 (10 microM), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 microM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 microM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 microM), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity.     

Excitatory synaptic currents in Purkinje cells

The N-methyl-D-aspartate (NMDA) and non-NMDA classes of glutamate receptor combine in many regions of the central nervous system to form a dual-component excitatory postsynaptic current. Non-NMDA receptors mediate synaptic transmission at the resting potential, whereas NMDA receptors contribute during periods of postsynaptic depolarization and play a role in the generation of long-term synaptic potentiation. To investigate the receptor types underlying excitatory synaptic transmission in the cerebellum, we have recorded excitatory postsynaptic currents (EPSCS), by using whole-cell techniques, from Purkinje cells in adult rat cerebellar slices. Stimulation in the white matter or granule-cell layer resulted in an all-or-none synaptic current as a result of climbing-fibre activation. Stimulation in the molecular layer caused a graded synaptic current, as expected for activation of parallel fibres. When the parallel fibres were stimulated twice at an interval of 40 ms, the second EPSC was facilitated; similar paired-pulse stimulation of the climbing fibre resulted in a depression of the second EPSC. Both parallel-fibre and climbing-fibre responses exhibited linear current-voltage relations. At a holding potential of -40 mV or in the nominal absence of Mg2+ these synaptic responses were unaffected by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). NMDA applied to the bath failed to evoke an inward current, whereas aspartate or glutamate induced a substantial current; this current was, however, largely reduced by CNQX, indicating that non-NMDA receptors mediate this response. These results indicate that both types of excitatory input to adult Purkinje cells are mediated exclusively by glutamate receptors of the non-NMDA type, and that these cells entirely lack NMDA receptors.     

Opposite modulation of capsaicin-evoked substance P release by glutamate receptors

Substance P and glutamate are present in primary afferent C-fibers and play important roles in persistent inflammatory and neuropathic pain. In the present study, we have examined whether activation of different glutamate receptor subtypes modulates the release of substance P evoked by the C-fiber selective stimulant capsaicin (1 μM) from rat trigeminal nucleus slices. The selective NMDA glutamate receptor agonist L-CCG-IV (1–10 μM) enhanced capsaicin-evoked substance P release about 100%. This facilitatory effect was blocked by 0.3 μM MK-801, a selective NMDA receptor antagonist. The metabotropic glutamate receptor agonists L-AP4 (group III) and DHPG (group I) (30–100 μM) inhibited capsaicin-evoked substance P release by approximately 60%. These inhibitory effects were blocked by the selective metabotropic glutamate receptor antagonist (±)-MCPG (5 μM). On the other hand, AMPA and kainate (0.1–10 μM), did not significantly affect capsaicin-evoked substance P release. Thus, substance P release from non-myelinated primary afferents, and possibly nociception, may be under the functional antagonistic control of some metabotropic and ionotropic glutamate receptor subtypes.     

Modulation of miniature inhibitory potentials in motoneurons of the turtle spinal cord by group II metabotropic glutamate receptors

The role of group II metabotropic glutamate receptors (mGluRs) in modulation of inhibitory synaptic activity was studied by intracellular recording of motoneuron miniature inhibitory spontaneous postsynaptic potentials (mIPSPs) in isolated lumbar segments of the turtle spinal cord in the medium containing TTX, CNQX, AP-5. The ratio of mIPSPs with fast and slow kinetics (83% vs 17%) is in accordance with the ratio shown for glycine- and GABA-mediated IPSP or IPSCs (Jones et al., 1988; Gao et al., 2001). In the majority of investigated motoneurons, the selective group II mGluRs antagonist EGLU (100-250 microM) increased the frequency of mIPSPs by 106.6 +/- 74.4% (n = 9) without affecting average amplitude, suggesting a presynaptic site of mGluRs action providing for the transmitter release reduction. The analysis of EGLU action on mIPSPs with different time courses (selection by half-width) showed that the frequency of inhancement of miniature inhibitory activity is caused by predominantly short-duration mIPSPs (ba 84.0 +/- 18.2%; n = 9), which are probably glycineergic. However, EGLU did not influence the mIPSPs frequency under condition of GABA-receptor blockade by bicuculline (20 microM). This fact suggest that group II mGluRs could modulate glycinergic transmission to the turtle spinal motoneurons on the necessary condition that GABergic system is active.     

Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor

l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.     

Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Long-term depression of mGluR1 signaling

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

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate Receptor Stimulation in Rat Primary Cortical Cultures

Abstract— Recent studies have identified at least two homologous mitogen-activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans -(±)-1-amino-1,3-cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)-α-(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N -methyl- d -aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABA_A receptor-mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

Novel 5-substituted 1-pyrazolol analogues of ibotenic acid: synthesis and pharmacology at glutamate receptors

5-Substituted 1-pyrazolol analogues of ibotenic acid have been synthesized and pharmacologically characterized on ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). The syntheses involved introduction of bromide, alkyls, phenyl and arylalkyls in the 5-position of 1-benzyloxypyrazole leading to 5-substituted (RS)-2-amino-(1-hydroxy-4-pyrazolyl)acetic acids (5a-l). The pharmacological activities of the synthesized analogues ranged from the 5-cyclopropylmethyl analogue (5f) with weak but selective affinity for NMDA receptors (IC(50)=35 microM), over the 5-n-propyl analogue (5c), which was a selective mGluR2 agonist (EC(50)=72 microM), to the 5-cyclohexylmethyl analogue (5g), which was a selective mGluR2 antagonist (K(i)=32 microM), and the 5-phenylethyl analogue (5j), which was a weak but apparently selective mGluR1 antagonist (K(i)=230 microM). This series of compounds afforded GluR ligands with a broad spectrum of pharmacological profiles, and showing potential for development of new compounds with subtype-selective activities at various GluRs.     

What mechanisms underlie involvement of different NMDA receptor subunits in the induction of hippocampal long-term potentiation and long-term depression?

There is a point of view that N-methyl-D-aspartate (NMDA) receptor subunit-specific signaling outcomes determine the direction of modifications of efficacy of synaptic transmission. Activation of NMDA receptors that contain the 2A subunit promotes LTP, while LTD requires activation of NMDA receptors containing 2B subunit. However, this hypothesis is inconsistent with some experimental data. For explanation of these data, we put forward an alternative hypothesis. According to this hypothesis, the activation of diverse subtypes of NMDA receptors can lead to ether LTP or LTD depending on the relation between posttetanic Ca2+ rise and increase in postsynaptic Ca2+ concentration produced by previous stimulation. Activation of NMDA receptors with 2B subunit can promote LTD of excitatory input to the pyramidal cell due to presence of these receptors on inhibitory interneurons, induction of the LTP in interneuron, and potentiation of inhibitory transmission between the interneuron and the target pyramidal cell.