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

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

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.     

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18‐1

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal‐regulated kinase (ERK) that mediates CB1R‐ and mGluR2/3‐induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock‐induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18‐1. Mimicking constitutive phosphorylation of Munc18‐1 results in a drastic decrease in synaptic transmission. ERK‐mediated phosphorylation of Munc18‐1 ultimately leads to degradation by the ubiquitin–proteasome system. Conversely, preventing ERK‐dependent Munc18‐1 phosphorylation increases synaptic strength. CB1R‐ and mGluR2/3‐induced synaptic inhibition and depolarization‐induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK‐dependent Munc18‐1 phosphorylation is blocked. Thus, ERK‐dependent Munc18‐1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.     

Supraspinal metabotropic glutamate receptor subtype 8: a switch to turn off pain

Glutamate is the main excitatory neurotransmitter in the central nervous system and as such controls the majority of synapses. Glutamatergic neurotransmission is mediated via ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). Signaling via mGluRs permits to finely tune, rather than turning on/off, the excitatory neurotransmission as the iGluRs do. Eight mGluRs (mGluR1-8) have been cloned so far, which have been divided into three groups based on sequence homology, pharmacological properties and second messenger signaling. mGluRs are widely expressed both on glia and neurons. On neurons they are located both at postsynaptic (group I) and presynaptic sites (group II and III). Group II and III mGluR stimulation reduces glutamate release, which can prove useful in pathological conditions characterized by elevated glutamatergic neurotransmission which include chronic pain. Indeed, mGluRs are widely distributed on pain neuraxis. The recent development of selective mGluR ligands has permitted investigating the individual role of each mGluR on pain control. The development of (S)-3,4-dicarboxyphenylglycine, a selective mGluR8 agonist, has revealed the mGluR8 role in inhibiting pain and its related affective consequences in chronic pain conditions. mGluR8 proved also to be overexpressed in pain controlling areas during pathological pain guaranteeing the availability of a switch for turning off abnormal pain. Thus, mGluR8 corresponds to an ideal target in designing novel analgesics. This review will focus on the novel insights into the mGluR8 role on pain control, with particular emphasis on the supraspinal descending pathway, an antinociceptive endogenous source, whose activation or disinhibition (via mGluR8) induces analgesia.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.     

Functional and molecular characterization of metabotropic glutamate receptors expressed in rat striatal cholinergic interneurones

In the present study we have used single-cell RT-PCR in conjunction with electrophysiology to examine the expression and functional properties of metabotropic glutamate receptors (mGluRs) expressed within biochemically identified cholinergic interneurones in the rat striatum. Using single-cell RT-PCR, it was possible to demonstrate the presence of mGluR1, mGluR2, mGluR3, mGluR5 and mGluR7 mRNAs within single cholinergic interneurones. Bath application of the non-selective mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) or the group-I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) depolarized all cholinergic neurones tested by activation of an inward current at -60 mV. The effects of DHPG were partially inhibited by the mGluR5 selective antagonist 6-methyl-2-(pherazo)-3-pyridinol and by the non-selective group-I antagonist alpha-methyl-4-carboxyphenylglycine but were not mimicked by the group-II and group-III selective mGluR agonists 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and L-2-amino-4-phosphonobutanoate (L-AP4), respectively. Intrastriatal stimulation evoked an excitatory postsynaptic current within cholinergic neurones that was reversibly inhibited by bath application of the group-II and group-III selective mGluR agonists DCG-IV and L-AP4, respectively, via presynaptic actions. In summary, we have identified the mGluRs expressed by striatal cholinergic interneurones and demonstrated that their activation produces modulatory effects via both pre- and postsynaptic mechanisms.     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

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 receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors

Metabotropic glutamate receptors (mGluRs) modulate important processes in cerebellum including long-term depression, which also requires formation of nitric oxide (NO) and cGMP. Some reports suggest that mGluRs could modulate the NO-cGMP pathway in cerebellum. However this modulation has not been studied in detail. The aim of this work was to assess by microdialysis in freely moving rats whether activation of mGluR5 modulates the NO-cGMP pathway in cerebellum in vivo and to analyze the underlying mechanisms. We show that mGluR5 activation increases extracellular glutamate, citrulline and cGMP in cerebellum. Blocking NMDA receptors with MK-801 does not prevent any of these effects, indicating that NMDA receptors activation is not required. However in the presence of MK-801 the effects are more transient, returning faster to basal levels. Blocking AMPA receptors prevents the increase in citrulline and cGMP induced by mGluR5 activation, but not the increase in glutamate. The release of glutamate is prevented by tetrodotoxin but not by fluoroacetate, indicating that glutamate is released from neurons and not from astrocytes. Activation of AMPA receptors increases citrulline and cGMP. These data indicate that activation of mGluR5 induces an increase of extracellular glutamate which activates AMPA receptors, leading to activation of nitric oxide synthase and increased NO, which activates guanylate cyclase, increasing cGMP. The response mediated by AMPA receptors desensitize rapidly. Activation of AMPA receptors also induces a mild depolarization, allowing activation of NMDA receptors which prolongs the duration of the effect initiated by activation of AMPA receptors. These data support that the three types of glutamate receptors: mGluR5, AMPA and NMDA cooperate in the modulation of the grade and duration of activation of the NO-cGMP pathway in cerebellum in vivo. This pathway would modulate cerebellar processes such as long-term depression.     

Fast and slow excitation of inhibitory cells in the CA3 region of the hippocampus

Pyramidal cells form excitatory synaptic connections with local inhibitory neurons in the hippocampus. This recurrent synapse plays a crucial stabilizing role in the control of hippocampal activity, since it transforms pyramidal cell population. Using a combination of dual recording from presynaptic and postsynaptic cells and anatomical techniques, we show that these synaptic connections often comprise a single site for liberation of excitatory transmitter. The resulting excitatory postsynaptic potentials (EPSCs) have a fast time course and a similar amplitude to miniature EPSCs recorded in tetrodotoxin and cobalt. In contrast, activation of metabotropic glutamate receptors (mGluRs) by transmitter liberated during repetitive activation of these synapses produces an excitation with a much slower time course. In addition to somatodendritic mGluRs, which excite inhibitory cells, a different species of mGluR is present on inhibitory cell terminals. This mGluR is activated by higher concentrations of the agonist t-1-amino-cyclopentyl–1,3-decarboxylate and acts to reduce γ-aminobutyric acid release. mGluRs, thus, have a dual action to enhance and to depress synaptic inhibition in the hippocampus. © 1995 John Wiley & Sons, Inc.     

Interaction between metabotropic glutamate receptor 7 and alpha tubulin

Metabotropic glutamate receptors (mGluRs) mediate a variety of responses to glutamate in the central nervous system. A primary role for group-III mGluRs is to inhibit neurotransmitter release from presynaptic terminals, but the molecular mechanisms that regulate presynaptic trafficking and activity of group-III mGluRs are not well understood. Here, we describe the interaction of mGluR7, a group-III mGluR and presynaptic autoreceptor, with the cytoskeletal protein, alpha tubulin. The mGluR7 carboxy terminal (CT) region was expressed as a GST fusion protein and incubated with rat brain extract to purify potential mGluR7-interacting proteins. These studies yielded a single prominent mGluR7 CT-associated protein of 55 kDa, which subsequent microsequencing analysis revealed to be alpha tubulin. Coimmunoprecipitation assays confirmed that full-length mGluR7 and alpha tubulin interact in rat brain as well as in BHK cells stably expressing mGluR7a, a splice variant of mGluR7. In addition, protein overlay experiments showed that the CT domain of mGluR7a binds specifically to purified tubulin and calmodulin, but not to bovine serum albumin. Further pull-down studies revealed that another splice variant mGluR7b also interacts with alpha tubulin, indicating that the binding region is not localized to the splice-variant regions of either mGluR7a (900-915) or mGluR7b (900-923). Indeed, deletion mutagenesis experiments revealed that the alpha tubulin-binding site is located within amino acids 873-892 of the mGluR7 CT domain, a region known to be important for regulation of mGluR7 trafficking. Interestingly, activation of mGluR7a in cells results in an immediate and significant decrease in alpha tubulin binding. These data suggest that the mGluR7/alpha tubulin interaction may provide a mechanism to control access of the CT domain to regulatory molecules, or alternatively, that this interaction may lead to morphological changes in the presynaptic membrane in response to receptor activation.     

Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils

LPS is an efficient sensitizer of the neutrophil exocytic response to a second stimulus. Although neutrophil exocytosis in response to pathogen-derived molecules plays an important role in the innate immune response to infections, the molecular mechanism underlying LPS-dependent regulation of neutrophil exocytosis is currently unknown. The small GTPase Rab27a and its effector Munc13-4 regulate exocytosis in hematopoietic cells. Whether Rab27a and Munc13-4 modulate discrete steps or the same steps during exocytosis also remains unknown. Here, using Munc13-4- and Rab27a-deficient neutrophils, we analyzed the mechanism of lipopolysaccharide-dependent vesicular priming to amplify exocytosis of azurophilic granules. We found that both Munc13-4 and Rab27a are necessary to mediate LPS-dependent priming of exocytosis. However, we show that LPS-induced mobilization of a small population of readily releasable vesicles is a Munc13-4-dependent but Rab27a-independent process. LPS-induced priming regulation could not be fully explained by secretory organelle maturation as the redistribution of the secretory proteins Rab27a or Munc13-4 in response to LPS treatment was minimal. Using total internal reflection fluorescence microscopy and a novel mouse model expressing EGFP-Rab27a under the endogenous Rab27a promoter but lacking Munc13-4, we demonstrate that Munc13-4 is essential for the mechanism of LPS-dependent exocytosis in neutrophils and unraveled a novel mechanism of vesicular dynamics in which Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis.     

Activation of Metabotropic Glutamate Receptors Inhibits Synapsin I Phosphorylation in Visceral Sensory Neurons

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca⁺⁺ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mm KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-α-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia. Received 28 June 2000/Revised: 11 September 2000     

Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.     

Doc2 alpha and Munc13-4 regulate Ca(2+) -dependent secretory lysosome exocytosis in mast cells

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.     

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1-methyl-4-phenylpyridinium (MPP+) astroglial conditioned medium-induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.     

mGluRs Modulate Strength and Timing of Excitatory Transmission in Hippocampal Area CA3

Excitatory transmission within hippocampal area CA3 stems from three major glutamatergic pathways: the perforant path formed by axons of layer II stellate cells in the entorhinal cortex, the mossy fiber axons originating from the dentate gyrus granule cells, and the recurrent axon collaterals of CA3 pyramidal cells. The synaptic communication of each of these pathways is modulated by metabotropic glutamate receptors that fine-tune the signal by affecting both the timing and strength of the connection. Within area CA3 of the hippocampus, group I mGluRs (mGluR1 and mGluR5) are expressed postsynaptically, whereas group II (mGluR2 and mGluR3) and III mGluRs (mGluR4, mGluR7, and mGluR8) are expressed presynaptically. Receptors from each group have been demonstrated to be required for different forms of pre- and postsynaptic long-term plasticity and also have been implicated in regulating short-term plasticity. A recent observation has demonstrated that a presynaptically expressed mGluR can affect the timing of action potentials elicited in the postsynaptic target. Interestingly, mGluRs can be distributed in a target-specific manner, such that synaptic input from one presynaptic neuron can be modulated by different receptors at each of its postsynaptic targets. Consequently, mGluRs provide a mechanism for synaptic specialization of glutamatergic transmission in the hippocampus. This review will highlight the variability in mGluR modulation of excitatory transmission within area CA3 with an emphasis on how these receptors contribute to the strength and timing of network activity within pyramidal cells and interneurons.     

Group II Metabotropic Glutamate Receptors Depress Synaptic Transmission onto Subicular Burst Firing Neurons

The subiculum (SUB) is a pivotal structure positioned between the hippocampus proper and various cortical and subcortical areas. Despite the growing body of anatomical and intrinsic electrophysiological data of subicular neurons, modulation of synaptic transmission in the SUB is not well understood. In the present study we investigated the role of group II metabotropic glutamate receptors (mGluRs), which have been shown to be involved in the regulation of synaptic transmission by suppressing presynaptic cAMP activity. Using field potential and patch-clamp whole cell recordings we demonstrate that glutamatergic transmission at CA1-SUB synapses is depressed by group II mGluRs in a cell-type specific manner. Application of the group II mGluR agonist (2S,1′R,2′R,3′R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) led to a significantly higher reduction of excitatory postsynaptic currents in subicular bursting cells than in regular firing cells. We further used low-frequency stimulation protocols and brief high-frequency bursts to test whether synaptically released glutamate is capable of activating presynaptic mGluRs. However, neither frequency facilitation is enhanced in the presence of the group II mGluR antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495, nor is a test stimulus given after a high-frequency burst. In summary, we present pharmacological evidence for presynaptic group II mGluRs targeting subicular bursting cells, but both low- and high-frequency stimulation protocols failed to activate presynaptically located mGluRs.     

alphaCaMKII Is essential for cerebellar LTD and motor learning

Activation of postsynaptic alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) by calcium influx is a prerequisite for the induction of long-term potentiation (LTP) at most excitatory synapses in the hippocampus and cortex. Here we show that postsynaptic LTP is unaffected at parallel fiber-Purkinje cell synapses in the cerebellum of alphaCaMKII(-/-) mice. In contrast, a long-term depression (LTD) protocol resulted in only transient depression in juvenile alphaCaMKII(-/-) mutants and in robust potentiation in adult mutants. This suggests that the function of alphaCaMKII in parallel fiber-Purkinje cell plasticity is opposite to its function at excitatory hippocampal and cortical synapses. Furthermore, alphaCaMKII(-/-) mice showed impaired gain-increase adaptation of both the vestibular ocular reflex and optokinetic reflex. Since Purkinje cells are the only cells in the cerebellum that express alphaCaMKII, our data suggest that an impairment of parallel fiber LTD, while leaving LTP intact, is sufficient to disrupt this form of cerebellar learning.