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.     

Associative short-term synaptic plasticity mediated by endocannabinoids

Associative learning is important on rapid timescales, but no suitable form of short-term plasticity has been identified that is both associative and synapse specific. Here, we assess whether endocannabinoids can mediate such plasticity. In the cerebellum, bursts of parallel fiber (PF) activity evoke endocannabinoid release from Purkinje cell dendrites that results in retrograde synaptic inhibition lasting seconds. We find that the powerful climbing fiber (CF) to Purkinje cell synapse regulates this inhibition. Compared to PF stimulation alone, coactivation of PF and CF synapses greatly enhanced endocannabinoid-mediated inhibition of PF synapses. Retrograde inhibition was restricted to PFs activated within several hundred milliseconds of CF activation. This associative plasticity reflects two aspects of calcium-dependent endocannabinoid release. First, PF-mediated activation of metabotropic glutamate receptors locally reduced the dendritic calcium levels required for endocannabinoid release. Second, CF and PF coactivation evoked localized supralinear dendritic calcium signals. Thus, endocannabinoids mediate transient associative synaptic plasticity.     

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.     

Endocannabinoids control the induction of cerebellar LTD

The long-term depression (LTD) of parallel fiber (PF) synapses onto Purkinje cells plays a central role in motor learning. Endocannabinoid release and LTD induction both depend upon activation of the metabotropic glutamate receptor mGluR1, require postsynaptic calcium increases, are synapse specific, and have a similar dependence on the associative activation of PF and climbing fiber synapses. These similarities suggest that endocannabinoid release could account for many features of cerebellar LTD. Here we show that LTD induction is blocked by a cannabinoid receptor (CB1R) antagonist, by inhibiting the synthesis of the endocannabinoid 2-arachidonyl glycerol (2-AG), and is absent in mice lacking the CB1R. Although CB1Rs are prominently expressed presynaptically at PF synapses, LTD is expressed postsynaptically. In contrast, a previously described transient form of inhibition mediated by endocannabinoids is expressed presynaptically. This indicates that Purkinje cells release 2-AG that activates CB1Rs to both transiently inhibit release and induce a postsynaptic form of LTD.     

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.     

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.     

Long-term potentiation of neuronal glutamate transporters

Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.     

Climbing fiber innervation of NG2-expressing glia in the mammalian cerebellum

The molecular layer of the cerebellar cortex is populated by glial progenitors that express ionotropic glutamate receptors and extend numerous processes among Purkinje cell dendrites. Here, we show that release of glutamate from climbing fiber (CF) axons produces AMPA receptor currents with rapid kinetics in these NG2-immunoreactive glial cells (NG2+ cells) in cerebellar slices. NG2+ cells may receive up to 70 discrete inputs from one CF and, unlike mature Purkinje cells, are often innervated by multiple CFs. Paired Purkinje cell-NG2+ cell recordings show that one CF can innervate both cell types. CF boutons make direct synaptic junctions with NG2+ cell processes, indicating that this rapid neuron-glia signaling occurs at discrete sites rather than through ectopic release at CF-Purkinje cell synapses. This robust activation of Ca2+-permeable AMPA receptors in NG2+ cells expands the influence of the olivocerebellar projection to this abundant class of glial progenitors.     

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.     

Long-term plasticity mediated by mGluR1 at a retinal reciprocal synapse

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.     

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.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

Bidirectional parallel fiber plasticity in the cerebellum under climbing fiber control

Cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses can undergo postsynaptically expressed long-term depression (LTD) or long-term potentiation (LTP) depending on whether or not the climbing fiber (CF) input is coactivated during tetanization. Here, we show that modifications of the postsynaptic calcium load using the calcium chelator BAPTA or photolytic calcium uncaging result in a reversal of the expected polarity of synaptic gain change. At higher concentrations, BAPTA blocks PF-LTP. These data indicate that PF-LTD requires a higher calcium threshold amplitude than PF-LTP induction and suggest that CF activity acts as a polarity switch by providing dendritic calcium transients. Moreover, previous CF-LTD induction changes the relative PF-LTD versus -LTP induction probability. These findings suggest that bidirectional cerebellar learning is governed by a calcium threshold rule operating "inverse" to the mechanism previously described at other glutamatergic synapses (BCM rule) and that the LTD/LTP induction probability is under heterosynaptic climbing fiber control.     

Glutamate transporters bring competition to the synapse

Glutamate transporters (GluTs) prevent the accumulation of glutamate and influence the occupancy of receptors at synapses. The ability of extrasynaptic NMDA receptors and metabotropic glutamate receptors to participate in signaling is tightly regulated by GluT activity. Astrocytes express the highest density of GluTs and dominate clearance away from these receptors; synapses that are not associated with astrocyte processes experience greater mGluR activation and can be exposed to glutamate released at adjacent synapses. Although less abundant, neuronal transporters residing in the postsynaptic membrane can also shield receptors from the glutamate that is released. The diversity in synaptic morphology suggests a correspondingly rich diversity of GluT function in excitatory transmission.     

δ-Glutamate Receptors Are Differentially Distributed at Parallel and Climbing Fiber Synapses on Purkinje Cells

Abstract: Neurons containing multiple excitatory inputs may sort and target glutamate receptor subtypes to subsets of synapses. A good model for testing this hypothesis is the Purkinje cell, which expresses significant levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, kainate, N -methyl- d -aspartate, δ-, and metabotropic glutamate receptors. Purkinje cells receive two excitatory inputs, the parallel and climbing fibers; the combined effect of stimulation of these two inputs is to produce long-term depression of parallel fiber/Purkinje cell neurotransmission. Distribution of glutamate receptors in these two synapse populations in rat cerebella was studied using preembedding immunocytochemistry with antibodies to GluR1, GluR2/3, GluR5-7, NR1, δ1/2, and mGluR1α. Moderate/dense postsynaptic staining was most frequent in postsynaptic densities and spines of both parallel and climbing fiber synapses with mGluR1α antibody, was intermediate in frequency with GluR2/3 and GluR5-7 antibodies, and was least frequent with GluR1 and NR1 antibodies. The most striking finding was the absence of significant postsynaptic staining with δ1/2 antibody in climbing fiber synapses in adult animals, even though postsynaptic staining was prevalent in parallel fiber synapses with this antibody. In contrast to adults, moderate/dense postsynaptic immunolabeling of climbing fiber synapses with δ1/2 antibody was common in rats at 10 days postnatal. This study provides direct morphological evidence that δ-glutamate receptors are differentially targeted to synapse populations. Our results support previous suggestions that δ2 is involved in development of parallel and climbing fiber synapses and in long-term depression of parallel fiber/Purkinje synaptic responses in adults.     

Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.     

Endocannabinoids potentiate synaptic transmission through stimulation of astrocytes

Endocannabinoids and their receptor CB1 play key roles in brain function. Astrocytes express CB1Rs that are activated by endocannabinoids released by neurons. However, the consequences of the endocannabinoid-mediated neuron-astrocyte signaling on synaptic transmission are unknown. We show that endocannabinoids released by hippocampal pyramidal neurons increase the probability of transmitter release at CA3-CA1 synapses. This synaptic potentiation is due to CB1R-induced Ca(2+) elevations in astrocytes, which stimulate the release of glutamate that activates presynaptic metabotropic glutamate receptors. While endocannabinoids induce synaptic depression in the stimulated neuron by direct activation of presynaptic CB1Rs, they indirectly lead to synaptic potentiation in relatively more distant neurons by activation of CB1Rs in astrocytes. Hence, astrocyte calcium signal evoked by endogenous stimuli (neuron-released endocannabinoids) modulates synaptic transmission. Therefore, astrocytes respond to endocannabinoids that then potentiate synaptic transmission, indicating that astrocytes are actively involved in brain physiology.     

Roles of phospholipase Cbeta4 in synapse elimination and plasticity in developing and mature cerebellum

The beta isoforms of phospholipase C (PLCbetas) are thought to mediate signals from metabotropic glutamate receptor subtype 1 (mGluR1) that is crucial for the modulation of synaptic transmission and plasticity. Among four PLCbeta isoforms, PLCbeta4 is one of the two major isoforms expressed in cerebellar Purkinje cells. The authors have studied the roles of PLCbeta4 by analyzing PLCbeta4 knockout mice, which are viable, but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple climbing fiber innervation is clearly impaired in the rostral portion of the cerebellar vermis, where PLCbeta4 mRNA is predominantly expressed in the wild-type mice. In the adult, long-term depression is deficient at parallel fiber to Purkinje cell synapses in the rostral cerebellum of the PLCbeta4 knockout mice. The impairment of climbing fiber synapse elimination and the loss of long-term depression are similar to those seen in mice defective in mGluR1, Galphaq, or protein kinase C. Thus, the authors' results strongly suggest that PLCbeta4 is part of a signaling pathway, including the mGluR1, Galphaq and protein kinase C, which is crucial for both climbing fiber synapse elimination in the developing cerebellum and long-term depression induction in the mature cerebellum.     

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.     

Differential expression of posttetanic potentiation and retrograde signaling mediate target-dependent short-term synaptic plasticity

Short-term synaptic plasticity influences how presynaptic spike patterns control the firing of postsynaptic targets. Here we investigated whether specific mechanisms of short-term plasticity are regulated in a target-dependent manner by comparing synapses made by cerebellar granule cell parallel fibers onto Golgi cells (PF-->GC synapse) and Purkinje cells (PF-->PC synapse). Both synapses exhibited similar facilitation, suggesting that any differential short-term plasticity does not reflect differences in the initial release probability. PF-->PC synapses were highly sensitive to stimulus bursts, which could result in either depression of subsequent responses, mediated by endocannabinoid-dependent retrograde signaling, or enhancement of responses through posttetanic potentiation (PTP). In contrast, stimulus bursts had remarkably little effect on the strength of PF-->GC synapses. Unlike PCs, GCs were unable to regulate their PF synapses by releasing endocannabinoids. Moreover, PTP was reduced at the PF-->GC synapse compared to the PF-->PC synapse. Thus, the target-dependence of PF synapses arises from the differential expression of both retrograde signaling and PTP.