Stimulation Of Metabotropic Glutamate Receptors Research Articles

Generation of time delays: Simplified models of intracellular signalling in cerebellar Purkinje cells

In many neuronal systems, information is encoded in temporal spike patterns. The recognition and storage of temporal patterns requires the generation and modulation of time delays between inputs and outputs. In cerebellar Purkinje cells, stimulation of metabotropic glutamate receptors (mGluRs) results in a delayed calcium and voltage response that has been implicated in classical conditioning and temporal pattern recognition. Here, we analyse and simplify a complex model of the intracellular signalling network that has been proposed as a substrate for this delayed response. We systematically simplify the original model, present a minimal model of time delay generation, and show that a delayed response can be produced by the combination of negative feedback and autocatalysis, without any intervening signalling steps that would contribute additive delays. The minimal model is analysed using phase plane methods, and classified as an excitable system. We discuss the implication of excitability for computations performed by intracellular signalling networks in general.

Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine

The gliotransmitter D-serine is released upon (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and metabotropic glutamate receptor stimulation, but the mechanisms involved are unknown. Here, by using a highly sensitive bioassay to continuously monitor extracellular D-serine levels, we have investigated the pathways used in its release. We reveal that D-serine release is inhibited by removal of extracellular calcium and augmented by increasing extracellular calcium or after treatment with the Ca(2+) ionophore A23187. Furthermore, release of the amino acid is considerably reduced after depletion of thapsigargin-sensitive intracellular Ca(2+) stores or chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H(+)-ATPase inhibitor, indicating a role for the vesicular proton gradient in the transmitter storage/release. In addition, agonist-evoked D-serine release was sensitive to tetanus neurotoxin. Finally, immunocytochemical and sucrose density gradient analysis revealed that a large fraction of D-serine colocalized with synaptobrevin/VAMP2, suggesting that it is stored in VAMP2-bearing vesicles. In summary, our study reveals the cellular mechanisms subserving D-serine release and highlights the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine.

Repeated administration of AMPA or a metabotropic glutamate receptor agonist into the rat ventral tegmental area augments the subsequent behavioral hyperactivity induced by cocaine

Glutamate receptors and their related second messengers in the ventral tegmental area (VTA) are known to play critical roles in the initiation of behavioral sensitization to cocaine. To evaluate the hypothesis that repeated intra-VTA microinjections of the ionotropic glutamate agonist, AMPA, or the metabotropic glutamate agonist, t-ACPD, augment the behavioral hyperactivity induced by a subsequent challenge injection of cocaine. In addition, the dependency of the t-ACPD effect on activation of the calcium/calmodulin-dependent kinases (CaM-Ks) was assessed. Male Sprague-Dawley rats received four once-daily microinjections of saline, AMPA, t-ACPD, or t-ACPD plus the CaM-KII inhibitor KN-93 directly into the VTA; locomotor activity was measured for 120 min after each of the daily treatments. One week after the 4 treatment days, all animals received a challenge injection of cocaine (15 mg/kg, IP) and behavioral activity was monitored for 120 min. Intra-VTA administration of t-ACPD increased behavioral activity only on the first 2 treatment days, an effect that was blocked by pre-treatment with KN-93. Administration of AMPA into the VTA, in contrast, produced behavioral hyperactivity that sensitized over the 4 treatment days. Following the cocaine challenge injection, there was an augmentation of cocaine-induced behavioral hyperactivity in the groups pretreated with AMPA or t-ACPD but not in the animals administered t-ACPD plus KN-93. These results indicate that repeated stimulation of AMPA or metabotropic glutamate receptors in the VTA mimics the initiation of behavioral sensitization to cocaine. The present findings also suggest that glutamate agonist-induced activation of CaM-KII in the VTA plays a critical role in the behavioral and neuronal plasticity induced by repeated cocaine injections.

Effects of Metabotropic Glutamate Receptor Stimulation on Blood–Brain Barrier Permeability During Focal Cerebral Ischemia

This investigation was performed to evaluate whether ACPD [(1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid], a metabotropic glutamate receptor agonist, would enhance the degree of increase in blood-brain barrier (BBB) permeability caused by focal cerebral ischemia. In this study, male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, patches of 10(-5) M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min. Patches were changed every 10 min. One hour after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (Ki) of [alpha-14C] aminoisobutyric acid. There were no statistical differences in systemic blood pressures and heart rates between these groups. Blood gases were within normal limits. In the control group, the Ki of ischemic cortex (IC) was 2.1 times that of the contralateral cortex (CC) (3.7+/-0.9 vs. 1.8+/-0.3 microl/g/min). In the ACPD group, the Ki of the IC was 3.3 times that of the CC (5.0+/-0.7 vs. 1.5+/-0.4 microl/g/min). The increase in Ki of the ACPD group in the ischemic cortex was significantly greater than that in the control group. There was no significant difference in the Ki of the CC between these groups. Our data suggest that activation metabotropic glutamate receptors in the cortex can further augment the increase in BBB permeability caused by focal ischemia.

The metabotropic glutamate receptor 5 antagonist MPEP and the mGluR2 agonist LY379268 modify disease progression in a transgenic mouse model of Huntington's disease

Chronic glutamate mediated excitotoxicity has been suggested to contribute to the pathogenesis of Huntington's disease (HD). Both, inhibition of glutamate release through stimulation of presynaptic metabotropic glutamate receptor (mGluR) 2 and blockade of postsynaptic mGluR5 have been demonstrated to be neuroprotective against excitotoxicity. R6/2 HD transgenic mice which express an expanded CAG triplet repeat serve as a well-characterized mouse model for HD with progressing neurological abnormalities and limited survival. We treated R6/2 HD transgenic mice with either the mGluR2 agonist LY379268 (1.2 mg/kg) or with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (100 mg/kg) orally from a presymptomatic stage until death to investigate their potential disease modifying effects. We found that survival time in both the MPEP treated mice and the LY379268 treated mice was significantly increased in comparison to placebo treated transgenic controls (14.87±0.14 and 14.22±0.11 weeks versus 12.87±0.11 weeks, respectively). Additionally, the progressive decline in motor coordination of HD transgenic mice as tested with the rotarod test was significantly attenuated in MPEP- but not in LY379268-treated mice. Early pathological hyperactivity, which can be found in placebo treated HD transgenic mice, was significantly attenuated by both MPEP and LY379268 treatment. Immunohistologial examination of HD characteristic neuronal intranuclear inclusion (NII), however, demonstrated no effect on NII formation by either of the treatments applied. These data suggest that inhibition of glutamate neurotransmission via specific interaction with mGluRs might be interesting for both inhibition of disease progression as well as early symptomatic treatment in HD.

Activity-dependent Expression of Inositol 1,4,5-Trisphosphate Receptor Type 1 in Hippocampal Neurons

There are several lines of evidence showing that synaptic activity regulates the level of expression of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in neurons. In this study, we examined the effect of chronic activity blockade on the localization and level of IP3R1 expression in cultured hippocampal neurons. We found that chronic blockade of NMDA receptors (NMDARs), one of the major Ca(2+)-permeable ion channels, increased the number of neurons that express a high level of IP3R1 without any apparent changes in its intracellular localization. Interestingly, this up-regulation was time-dependent; there was no clear change in IP3R1 expression level up to day 5 of the NMDAR blockade, but expression increased at day 6, and the increased expression level persisted for at least a week. The up-regulation of IP3R1 depended on transcription and protein synthesis and required cAMP-dependent protein kinase activity. Moreover, although most of the control neurons did not respond to the metabotropic glutamate receptor (mGluR) stimulation, the 2-amino-5-phosphonopentanoic acid-treated neurons with high IP3R1 expression became sensitive to mGluR stimulation. Furthermore, we also found that hippocampal neurons transiently overexpressing green fluorescent protein-tagged IP3R1 released Ca2+ in response to mGluR and muscarinic acetylcholine receptor stimulation. These findings suggested that chronic NMDAR blockade increased the IP3R1 expression and enhanced sensitivity to mGluR stimulation. The change in IP3R1 expression level in response to alteration of synaptic activity may be an important determinant of the sensitivity of Ca2+ stores to G-protein-coupled receptor stimulation and would help to maintain intracellular Ca2+ homeostasis in hippocampal neurons.

Up-regulation of soluble amyloid precursor protein fragment secretion in the rat retina in vivo by metabotropic glutamate receptor stimulation

Using the novel rat retinal-vitreal model we have investigated the effect of metabotropic glutamate receptor activation on amyloid precursor protein (APP) metabolism. The release of low mol. wt fragments of APP, at 15-23 kDa in particular, was markedly up-regulated by the metabotropic glutamate receptor agonist (1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid ((1S,3R)-ACPD) in a concentration- and time-dependent manner, and this response was blocked by the receptor antagonist (S)-alpha-methyl-4-caboxyphenylglycine ((S)-MCPG). These results, together with the observation of a lack of deleterious effects of (1S,3R)-ACPD on the retinal neurons, support a physiological role of metabotropic glutamate receptors in mediating the release of soluble APP fragments, an action which may have important functional and therapeutic implications for Alzheimer's disease.

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus.

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC50 of 0.19 +/- 0.05 microM. DCG IV (1 microM) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 nM) and mimicked by another selective group II agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Inhibition of EPSC amplitude by DCG IV and L-CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 microM) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 microM), by the adenylyl cyclase inhibitor MDL 12230A (20 microM), or by the phosphodiesterase inhibitor Ro 20-1724 (50 microM). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 microM), H-8 (50 microM) and HA-1004 (100 microM). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.

Effects of metabotropic glutamate receptor stimulation on cerebral O2 consumption and blood flow during focal cerebral ischemia in rats.

This investigation was performed to evaluate the effects of ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O2 consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10(-5) M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C14-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 +/- 11 vs. 110 +/- 11 ml/min/100 g, O2 consumption: 2.9 +/- 0.4 vs. 5.4 +/- 1.1 ml O2/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 +/- 0.2 vs. 6.9 +/- 0.3 ml O2/100 ml) and oxygen consumption of the IC (4.3 +/- 1.5 vs. 2.9 +/- 0.4) compared to the control IC. Our data demonstrated that topical application of 10(-5) M ACPD to the ischemic area worsened cerebral O2 balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.

A Receptor for Activated C Kinase Is Part of Messenger Ribonucleoprotein Complexes Associated with PolyA-mRNAs in Neurons

Long-lasting changes in synaptic functions after an appropriate stimulus require altered protein expression at the synapse. To restrict changes in protein composition to activated synapses, proteins may be synthesized locally as a result of transmitter receptor-triggered signaling pathways. Second messenger-controlled mechanisms that affect mRNA translation are essentially unknown. Here we report that a receptor for activated C kinase, RACK1, is a component of messenger ribonucleoprotein (mRNP) complexes. RACK1 is predominantly associated with polysome-bound, polyA-mRNAs that are being actively translated. We find it to be present in a complex with beta-tubulin and at least two mRNA-binding proteins, polyA-binding protein 1 and a 130 kDa polyA-mRNA binding protein (KIAA0217). Activation of PKCbeta2 in vitro by phosphatidylserine/diacylglycerol or in hippocampal slices by metabotropic glutamate receptor stimulation increased the amount of RACK1/PKCbeta2 associated with polysome-bound polyA-mRNAs. In vitro, PKCbeta2 can phosphorylate a subset of polyA-mRNA-associated proteins that are also phosphorylated under in vivo conditions. On the basis of these findings plus the somatodendritic localization of RACK1, we hypothesize that metabotropic glutamate receptor-triggered binding of activated PKCbeta2 to mRNP complexes bound to polyA-mRNAs is involved in activity-triggered control of protein synthesis.

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow during Focal Cerebral Ischemia in Rats

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow during Focal Cerebral Ischemia in Rats

The metabotropic glutamate receptor agonist 1 S,3 R-ACPD stimulates and modulates NMDA receptor mediated excitotoxicity in organotypic hippocampal slice cultures

The potential toxic effects of the metabotropic glutamate receptor agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and its interactions with the N-methyl- d-aspartate (NMDA) receptor were studied in hippocampal brain slice cultures, using densitometric measurements of the cellular uptake of propidium iodide (PI) to quantify neuronal degeneration. Cultures exposed to ACPD, showed a concentration (2–5 mM) and time (1–4 days) dependent increase in PI uptake in CA1, CA3 and dentate subfields after 24 h and 48 h of exposure, with CA1 pyramidal cells being most sensitive. The neurodegeneration induced by 2 mM ACPD was completely abolished by addition of 10 μM of the NMDA receptor antagonist (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801), while 20 μM of the 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[ f]quinoxaline-7-sulfonamide (NBQX) had no effect. Co-exposing cultures to a subtoxic dose of 300 μM ACPD together with 10 μM NMDA, which at this dose is known to induce a fairly selective degeneration of CA1 pyramidal cells, significantly increased the PI uptake in both CA1 and CA3, compared to cultures exposed to 10 μM NMDA only. Adding the 300 μM ACPD as pretreatment for 30 min followed by a 30 min wash in normal medium before the ACPD/NMDA co-exposure, eliminated the potentiation of NMDA toxicity. The potentiation was also blocked by addition of 10 or 100 μM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 μM 7-(hydroxyimino)cyclopropa[ b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (mGluR1 antagonist) had no effect. We conclude that, stimulation of metabotropic glutamate receptors with ACPD at concentrations of 2 mM or higher induces a distinct subfield-related and time and concentration dependent pattern of hippocampal degeneration, and that ACPD at subtoxic concentrations modulates NMDA-induced excitotoxicity through the mGluR5 receptor in a time dependent way.

Assembly of SNARE Core Complexes Prior to Neurotransmitter Release Sets the Readily Releasable Pool of Synaptic Vesicles

Precise regulation of neurotransmitter release is essential for the normal function of neural networks, but the mechanisms involved are largely unclear. Using superfused synaptosomes, we have studied the readily releasable pool of synaptic vesicles, measured as the amount of release triggered by hypertonic sucrose. We show that activation of presynaptic metabotropic glutamate receptors by dihydroxyphenylglycine and stimulation of protein kinase C by phorbol esters enhance the readily releasable pool of glutamate. Although the molecular nature of the readily releasable pool is unknown, one possibility is that during its generation, SNARE proteins form full core complexes, and that core complex formation occurs prior to neurotransmitter release. To test this possibility, we employed N-ethylmaleimide (NEM), an inhibitor of the ATPase N-ethylmaleimide-sensitive factor that dissociates core complexes, to study the relation of the readily releasable pool to core complex assembly in synaptosomes. NEM induced a dose-dependent increase in the readily releasable pool of neurotransmitters but by itself did not trigger release. Direct measurements of core complexes confirmed that NEM caused an increase in the levels of SNARE core complexes under these conditions. Our data suggest that in the readily releasable pool of synaptic vesicles, SNARE proteins are fully assembled into core complexes, and that SNARE complex assembly is a target of presynaptic regulation.

Hypoxic modulation of L-type Ca 2+ channels in inspiratory brainstem neurones: Intracellular signalling pathways and metabotropic glutamate receptors

Brief hypoxia (2 min) enhances the activity of L-type Ca 2+ (Ca L) channels. The effect is due to glutamate release and concomitant stimulation of metabotropic glutamate receptors of the mGLUR1/5 type [22] [S.L. Mironov, D.W. Richter, L-type Ca 2+ channels in inspiratory neurones and their modulation by hypoxia, J. Physiol. 512 (1998) 75–87.]. Besides increasing single channel activity, hypoxia induces a negative shift of the activation curve and slows down the inactivation of the Ca L current. In the present study we investigated these effects further, aiming to reveal intracellular signalling pathways that mediate the coupling between mGLURs and Ca L channels. Channel activity was recorded in cell-attached patches from inspiratory brainstem neurones of neonatal mice (P6–11). Ca L channels were inhibited by the mGluR2/3 agonists. mGluR1/5 agonists accelerated and mGluR2/3 agonists suppressed the respiratory output, and correspondingly modified the hypoxic response of the respiratory center. Ca L channels were also modulated by protein kinase C, but this did not prevent the hypoxic modification of channel activity. G-protein activators enhanced and G-protein inhibitors suppressed the Ca L channel activity, and in the presence of these agents the effects of hypoxia were abolished. Ryanodine but not thapsigargin inhibited the channel activity and occluded the hypoxic potentiation. Only G-protein-specific agents and ryanodine prevented the slowing down of inactivation induced by hypoxia. Our data indicate that coupling between mGluR1/5 and Ca L channels is mediated by pathways that utilize G-proteins and ryanodine receptors. Glutamate release and concomitant activation of Ca L channels are responsible for accelerating of respiratory rhythm during early hypoxia.

Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex

It is well documented that the descending endogenous analgesia system, including the periaqueductal gray (PAG) and the rostral ventral medulla (RVM), play an important role in modulation of nociceptive transmission and morphine- and cannabinoid-produced analgesia. Neurons in the PAG receive inputs from different nuclei of higher structures, including the anterior cingulate cortex (ACC). However, it is unclear if stimulation of neurons in the ACC modulates spinal nociceptive transmission. The present study has examined the effects of electrical stimulation and chemical activation of metabotropic glutamate receptors (mGluRs) in the ACC on a spinal nociceptive tail-flick (TF) reflex induced by noxious heating. Activation of the ACC at high intensities (up to 500 μA) of electrical stimulation did not produce any antinociceptive effect. Instead, at most sites within the ACC (n = 36 of 41 sites), electrical stimulation produced significant facilitation of the TF reflex (i.e. decreases in TF latency). Chemical activation of mGluRs within the ACC also produced a facilitatory effect. Descending facilitation from the ACC apparently relays at the RVM. Electrical stimulation in the RVM produces a biphasic modulatory effect, showing facilitation at low intensities and inhibition at higher intensities. The present study provides evidence that activation of mGluRs within the ACC can facilitate spinal nociception. Copyright 2000 European Federation of Chapters of the International Association for the Study of Pain

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ.

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.

Intrasubthalamic nucleus metabotropic glutamate receptor activation: a behavioral, Fos immunohistochemical and [ 14C]2-deoxyglucose autoradiographic study

Metabotropic glutamate receptors are a major class of excitatory amino acid receptors. Eight metabotropic glutamate receptor subtypes have been cloned, and are classified into three groups (I, II and III) based on amino acid sequence identity, effector systems and pharmacological profile. Previous results have shown that unilateral stimulation of metabotropic glutamate receptors in the subthalamic nucleus with the non-subtype-selective metabotropic glutamate receptor agonist 1 S,3 R-1-amino-1,3-cyclopentane dicarboxylate results in contralateral rotation in rats and Fos expression in the subthalamic nucleus. This suggests that metabotropic glutamate receptor stimulation results in altered subthalamic nucleus activity with consequent altered basal ganglia activity on the injected side. We sought to determine the metabotropic glutamate receptor subtype(s) involved and the functional neuroanatomy underlying the rotational behavior. Unilateral intrasubthalamic nucleus injection of group II or group III metabotropic glutamate receptor agonists induced contralateral rotation. In addition to producing rotation, group II and group III metabotropic glutamate receptor agonists induce toxicity in the subthalamic nucleus and overlying thalamus. Following group II or group III subthalamic nucleus metabotropic glutamate receptor stimulation, there is Fos-like immunoreactivity in the globus pallidus, subthalamic nucleus, substantia nigra pars reticulata and entopeduncular nucleus, suggesting altered activity in subthalamic nucleus target regions. However, examination of [ 14C]2-deoxyglucose uptake suggests that the alterations in basal ganglia activity are different following group II versus group III metabotropic glutamate receptor stimulation, suggesting that rotation is occurring via different mechanisms. It appears that stimulation of subthalamic nucleus group II metabotropic glutamate receptors induces rotation by increasing subthalamic nucleus activity. These results suggest that group II metabotropic glutamate receptor antagonists may be useful for alleviating subthalamic nucleus overactivity in Parkinson's disease.

Ca2+ store-dependent potentiation of Ca2+-activated non-selective cation channels in rat hippocampal neurones in vitro.

1. Potentiation of calcium-activated non-selective cation (CAN) channels was studied in rat hippocampal neurones. CAN channels were activated by IP3-dependent Ca2+ release following metabotropic glutamate receptor (mGluR) stimulation either by Schaffer collateral input to CA1 neurones in brain slices in which ionotropic glutamate and GABAA receptors, K+ channels, and the Na+-Ca2+ exchanger were blocked or by application of the mGluR antagonist ACPD in cultured hippocampal neurones. 2. The CAN channel-dependent depolarization (DeltaVCAN) was potentiated when [Ca2+]i was increased in neurones impaled with Ca2+-containing microelectrodes. 3. Fura-2 measurements revealed a biphasic increase in [Ca2+]i when 200 microM ACPD was bath applied to cultured hippocampal neurones. This increase was greatly attenuated in the presence of Cd2+. 4. Thapsigargin (1 microM) caused marked potentiation of DeltaVCAN in CA1 neurones in the slices and of the CAN current (ICAN) measured in whole cell-clamped cultured hippocampal neurones. 5. Ryanodine (20 microM) also led to a potentiation of DeltaVCAN while neurones pretreated with 100 microM dantrolene failed to show potentiation of DeltaVCAN when impaled with Ca2+-containing microelectrodes. 6. The mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide m-chlorophenyl hydrazone (2 microM) also caused a potentiation of DeltaVCAN. 7. CAN channels are subject to considerable potentiation following an increase in [Ca2+]i due to Ca2+ release from IP3-sensitive, Ca2+-sensitive, or mitochondrial Ca2+ stores. This ICAN potentiation may play a crucial role in the 'amplification' phase of excitotoxicity.

Reversal of glutamate excitotoxicity by activation of PKC-associated metabotropic glutamate receptors in cerebellar granule cells relies on NR2C subunit expression.

Stimulation of metabotropic glutamate receptors (mGluRs) belonging to group I has been found to reduce N-methyl-D-aspartate (NMDA) receptor function in terms of both intracellular calcium concentration ([Ca2+]i) rise and neurotoxicity in cultured cerebellar granule cells. In the present study, we investigated whether the mGluR-elicited modulation of glutamate responses might rely on the heteromeric composition of NMDA receptor channel. NMDA receptors consist of two distinct groups of subunits: NR1, that is ubiquitously in the receptor complexes; and NR2A-D, that differentiate and potentiate NMDA receptor responses by assembling with NR1. Among NR2 subunits, only NR2A and NR2C mRNAs and relative proteins are detected in cerebellar granule cells at 10 days in vitro. To dissect the involvement of the two different subunits in making the NMDA receptor channel sensitive to modulation by group I mGluR agonists, expression of the NR2C subunit was prevented by treating the cells with specific antisense oligodeoxynucleotide (ODN). The capability of the mGluR agonists, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD, 100 microM) or 3 hydroxyphenylglycine (3HPG, 100 microM), and the protein kinase C (PKC) activator, 4beta-phorbol-12,13-dibutyrate (PDBu, 1 microM), to inhibit the function of resultant NMDA receptors was then evaluated. We found that depletion of the NR2C subunit abolished the inhibitory effect of group I mGluR stimulation on glutamate-induced [Ca2+]i rise and neurotoxicity. The antisense ODN treatment also prevented the inhibitory effect of PDBu on glutamate responses. Conversely, in NR2C-lacking neurons, both group I mGluRs and PKC stimulation enhanced NMDA receptor-mediated effects. The present findings indicate that the capability of PKC-associated mGluRs to modulate native NMDA receptor function relies on the heteromeric configuration of the receptor-channel complex. Particularly, expression of the NR2C subunit is required to make the NMDA receptor sensitive to inhibitory modulation by mGluRs or PKC activation.

Synaptic synthesis of the Fragile X protein: possible involvement in synapse maturation and elimination.

Fragile X mental retardation syndrome results from the absence of or a defect in the protein (FMRP) encoded by the FMR1 gene. FMRP is found in dendrites and synapses as well as in the neuronal cell soma and nucleus, and although it is known to bind to RNA, the function of the protein in neurons is not known. We have studied activity-dependent changes in postsynaptically localized protein translation in central nervous system neurons. We find that FMRP is one of the proteins produced at synapses following stimulation of metabotropic glutamate receptors. We have also observed that Fragile X knockout mice, like human Fragile X patients, have excess numbers of long, thin, immature-appearing dendritic processes. Together, these findings suggest that FMRP plays a role in the process whereby synaptic activity during development results in structural and functional maturation of the synapse. We hypothesize that synaptic synthesis of FMRP may be essential for activity-based synapse maturation and elimination, a key process in normal brain development.