Parallel fiber-Purkinje Cell Synapses Research Articles

High Transgene Expression by Lentiviral Vectors Causes Maldevelopment of Purkinje Cells In Vivo

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.

Adenosine signalling at immature parallel fibre–Purkinje cell synapses in rat cerebellum

The purine adenosine is an extracellular signalling molecule involved in a large number of physiological and pathological conditions throughout the mammalian brain. However little is known about how adenosine release and its subsequent clearance change during brain development. We have combined electrophysiology and microelectrode biosensor measurements to investigate the properties of adenosine signalling at early stages of cerebellar development, when parallel fibre-Purkinje cell synapses have recently been formed (postnatal days 9-12). At this stage of development, we could detect little or no inhibitory A(1) receptor tone in basal conditions and during trains of stimuli. Addition of pharmacological agents, to inhibit adenosine clearance, had only minor effects on synaptic transmission suggesting that under basal conditions, the concentration of adenosine moving in and out of the extracellular space is small. Active adenosine release was stimulated with hypoxia and trains of electrical stimuli. Although hypoxia released significant concentrations of adenosine, the release was delayed and slow. No adenosine release could be detected following electrical stimulation in the molecular layer. In conclusion, at this stage of development, although adenosine receptors and the mechanisms of adenosine clearance are present there is very little adenosine release.

Enhanced homosynaptic LTD in cerebellar Purkinje cells of the dystrophic MDX mouse

The purpose was to study homosynaptic long-term depression (LTD) at the parallel fiber-Purkinje cell synapse in the mdx mouse, a murine model of the human dystrophinopathy, Duchenne muscular dystrophy (DMD), in order to examine whether the absence of dystrophin affects the induction and extent of this form of synaptic plasticity. Sharp intracellular electrodes were used to record electrically evoked excitatory postsynaptic potentials (EPSPs) from identified Purkinje cells in cerebellar slices. The early phase of homosynaptic LTD, 7-16 min postinduction, was the same in mdx and wildtype Purkinje cells; however, the late phase of LTD, 35-44 min, was significantly enhanced in mdx Purkinje cells. We hypothesize that this enhancement of the late phase of homosynaptic LTD may be due to a disruption of Ca(2+) homeostasis associated with the absence of the protein dystrophin. These findings may explain some of the central nervous system deficiencies reported in DMD boys.

Presynaptic NR2A-containing NMDA receptors implement a high-pass filter synaptic plasticity rule

The detailed characterization of synaptic plasticity has led to the replacement of simple Hebbian rules by more complex rules depending on the order of presynaptic and postsynaptic action potentials. Here, we describe a mechanism endowing a plasticity rule with additional computational complexity--a dependence on the pattern of presynaptic action potentials. The classical Hebbian rule is based on detection of conjunctive presynaptic and postsynaptic activity by postsynaptic NMDA receptors, but there is also accumulating evidence for the existence of presynaptic NMDA receptors in several brain structures. Here, we examine the role of presynaptic NMDA receptors in defining the temporal structure of the plasticity rule governing induction of long-term depression (LTD) at the cerebellar parallel fiber-Purkinje cell synapse. We show that multiple presynaptic action potentials at frequencies between 40 Hz and 1 kHz are necessary for LTD induction. We characterize the subtype, kinetics, and role of presynaptic NMDA receptors involved in the induction of LTD, showing how the kinetics of the NR2A subunits expressed by parallel fibers implement a high-pass filter plasticity rule that will selectively attenuate synapses undergoing high-frequency bursts of activity. Depending on the type of NMDA receptor subunit expressed, high-pass filters of different corner frequencies could be implemented at other synapses expressing NMDA autoreceptors.

Role of Presynaptic Kainate Receptors at Parallel Fiber–Purkinje Cell Synapses in Induction of Cerebellar LTD: Interplay With Climbing Fiber Input

Until recently, except for A1 adenosine, N-methyl-d-aspartate, and cannabinoid receptors, little effort has been made to unravel possible roles of parallel fiber (PF) presynaptic receptors in long-term depression (LTD) of synaptic transmission at PF-Purkinje cell (PC) synapses. Presynaptic kainate (KA) receptors are also present on PFs and might also influence LTD induction by modulating glutamate (Glu) release at PF-PC synapses. This hypothesis was tested by comparing the efficacy of two pairing protocols in inducing LTD in adult wild-type and knock-out mice for the Glu receptor 6 (GluR6) subunit of KA receptors. Activation of presynaptic KA receptors was unnecessary for LTD induction when PF inputs were paired with climbing fiber (CF) stimulation but became crucial when CF input was replaced by direct depolarization of PCs. By comparing basal paired-pulse facilitation of PF-excitatory postsynaptic currents (EPSCs) and depolarization-induced suppression of excitation in adult wild-type and GluR6 knock-out mice, it was shown that the participation of PF presynaptic KA receptors in LTD induction was likely to mainly result from their tonic activation by basal extracellular Glu, rather than from their activation by retrograde release of Glu by PCs during pairing protocols. Finally, this study suggests that, in adult mice, CFs not only participate in LTD induction by depolarizing postsynaptic cells but also by activating postsynaptic mGluR1alpha metabotropic glutamate receptors at CF-PC synapses.

βCaMKII controls the direction of plasticity at parallel fiber–Purkinje cell synapses

We found that betaCaMKII, the predominant CaMKII isoform of the cerebellum, is important for controlling the direction of plasticity at the parallel fiber-Purkinje cell synapse; a protocol that induced synaptic depression in wild-type mice resulted in synaptic potentiation in Camk2b knockout mice and vice versa. These findings provide us with unique experimental insight into the mechanisms that transduce graded calcium signals into either synaptic depression or potentiation.

Not glutamate but endocannabinoids mediate retrograde suppression of cerebellar parallel fiber to Purkinje cell synaptic transmission in young adult rodents

In the cerebellum of juvenile mice or rats, endocannabinoids are shown to mediate depolarization-induced suppression of excitation (DSE) and retrograde suppression induced by activation of type 1 metabotropic glutamate receptor (mGluR1) at parallel fiber (PF) to Purkinje cell (PC) synapses. However, recent studies showed that glutamate also mediated retrograde signaling through presynaptic kainate receptors in the cerebellum of young adult mice and rats. We reexamined this possibility in C57BL/6 mice at postnatal day 20–35 (P20–P35) and in Sprague–Dawley rats at P18–P24. We found that DSE at PF–PC synapses was abolished by AM251, a cannabinoid receptor antagonist, and by tetrahydrolipstatin (THL), a blocker of diacylglycerol lipase (DGL) that produces an endocannabinoid, 2-arachidonoylglycerol (2-AG). AM251 and THL did not affect depolarization-induced Ca 2+ transients in PCs, and THL did not suppress cannabinoid sensitivity of PFs. Moreover, DSE at PF–PC synapses was absent in CB 1 knockout mice. AM251 also eliminated transient suppression of PF–PC synaptic transmission following a brief burst of PF stimulation, a phenomenon known to be mediated by mGluR1. These results suggest that DSE and mGluR1-mediated suppression in young adult PCs are mediated by endocannabinoids, and that glutamate, if any, has little contribution.

Bistable Switches for Synaptic Plasticity

A persistent decrease in synaptic efficacy, called long-term depression (LTD), of the parallel fiber-Purkinje cell synapse is thought to underlie some forms of learning and memory in the cerebellum. Simulation studies predicted that mitogen-activated protein kinase and protein kinase C would mutually activate each other and make a bistable positive feedback loop, thereby providing a molecular basis for LTD. This claim stimulated experimenters to successfully demonstrate the feedback loop and its pivotal role in cerebellar LTD.

Cbln1 accumulates and colocalizes with Cbln3 and GluRδ2 at parallel fiber–Purkinje cell synapses in the mouse cerebellum

Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRdelta2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRdelta2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRdelta2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.

Cbln1 binds to specific postsynaptic sites at parallel fiber–Purkinje cell synapses in the cerebellum

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.

A Mutant δ2 Ionotropic Glutamate Receptor Exhibits Dual Regulation by Phosphoinositides

The δ2 glutamate receptor (GluRδ2) is considered a member of the ionotropic glutamate receptor family, although a specific ligand that activates the wild-type receptor has yet to be identified. GluRδ2 is enriched in the parallel fiber-Purkinje cell (PF-PC) synapse, but the precise physiological role of the receptor is still unclear. A naturally-occurring single point mutant in the third transmembrane domain of the receptor (A654T), named Lurcher (GluRδ2Lc), exhibits constitutive activity.Our previous preliminary results suggested that the δ2 glutamate receptor - Lurcher mutant is inhibited by direct interactions with phosphatidylinositol 4,5-bisphosphate (PIP2). Here we show that pre-incubation with wortmannin affects the activity of the receptor in a concentration-dependent manner, leading to an inhibition of the channel in the low micromolar range. This suggests that phosphatidylinositol 3-kinase (PI3K) could also be involved in the regulation of the δ2 glutamate receptor. We further investigate the involvement of phosphoinositides in the regulation of δ2 glutamate receptor using more specific PI3K and PI4K inhibitors as well as soluble stereoisomers of different phosphoinositides.

The interaction of Purkinje cell and Inhibitory Interneuron plasticity during classical conditioning

Event Abstract Back to Event The interaction of Purkinje cell and Inhibitory Interneuron plasticity during classical conditioning Decades of experimental work using the the eye-blink conditioning paradigm points to the Cerebellar Cortex (CC) as the locus of the motor conditioning engram. It is in the cerebellar cortex where a memory trace is formed that allows both to associate Conditioning (CS) and Unconditioned Stimulus (US) and to elicit an adaptively timed Conditioned Response (CR). Single cell recordings show that the evolution of the firing rate of a Purkinje (PU) cell during the CS-US period provides a substrate for such learning (Jirenhed et al., 2007). Classical computational theories of the Cerebellum (like the Marr-Albus-Ito model) predicted that the mechanism responsible for motor learning was plasticity at the parallel fiber to PU (pf-PU) synapse. However in vitro and in vivo studies have reported multiple loci of plasticity in the CC among them plasticity at the parallel fibers to Inhibitory Interneuron (pf-II) synapse (Jörntell, & Ekerot, 2002). This site of plasticity is of particular interest because it is inversely related to the synaptic dynamics of the pf-PU synapse: whenever the pf-PU synapse undergoes LTD the pf-II undergoes LTP, and vice versa. Hence, this suggests that the engram is not restricted to the parallel fiber Purkinje cell synapse only. A particular challenge for models that exclusive rely on the plasticity at pf-PU synapses is that they have difficulty to explain results that have shown that increasing CS intensity shortens the latency of already acquired CRs (Svensson et al, 1997). We hypothesize that the inclusion of the active role of the Inhibitory Interneurons (II) in learning can account for this CS intensity effect. We have implemented a biologically constrained model aimed at reproducing the learning process of a single Purkinje cell, in such a way that it will respond to presentations of a CS with an adaptively timed pause. In previous work we demonstrated that having plasticity at the pf-PU was sufficient to achieve this goal provided the cortico-nuclear-olivary negative feedback loop was included (Verschure and Mintz, 2000; Hofstötter et al., 2002). Now, we have extended this model and also included plasticity at the pf-II synapse in order to test its computational implications. Our results confirm that potentiation of the II can account for the CS intensity effect. Our model also predicts that if these two plasticity processes coexist it should be possible to identify different components in the dynamics of the acquisition/extinction processes at the level of single Purkinje cells. Although two components have already been founded in behavioral data (Ivarsson & Svensson, 2000) a confirmation of this prediction could only be obtained by analyzing the responses of single Purkinje cells in such experiments.

Climbing fiber signaling and cerebellar gain control

The physiology of climbing fiber signals in cerebellar Purkinje cells has been studied since the early days of electrophysiology. Both the climbing fiber-evoked complex spike and the role of climbing fiber activity in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses have become hallmark features of cerebellar physiology. However, the key role of climbing fiber signaling in cerebellar motor learning has been challenged by recent reports of forms of synaptic and non-synaptic plasticity in the cerebellar cortex that do not involve climbing fiber activity, but might well play a role in cerebellar learning. Moreover, cerebellar LTD does not seem to strictly require climbing fiber activity. These observations make it necessary to re-evaluate the role of climbing fiber signaling in cerebellar function. Here, we argue that climbing fiber signaling is about adjusting relative probabilities for the induction of LTD and long-term potentiation (LTP) at parallel fiber synapses. Complex spike-associated, dendritic calcium transients control postsynaptic LTD and LTP induction. High calcium transients, provided by complex spike activity, do not only favor postsynaptic LTD induction, but simultaneously trigger retrograde cannabinoid signaling, which blocks the induction of presynaptic LTP. Plasticity of the climbing fiber input itself provides additional means to fine-tune complex spike associated calcium signaling and thus to adjust the gain of heterosynaptic climbing fiber control. In addition to dendritic calcium transients, climbing fiber activity leads to the release of the neuropeptide corticotropin-releasing factor (CRF), which facilitates LTD induction at both parallel fiber and climbing fiber synapses.

Reduction of intracellular Ca 2+ requirement for induction of bidirectional synaptic plasticity at parallel fiber-Purkinje cell synapses in mice lacking delphilin

Reduction of intracellular Ca 2+ requirement for induction of bidirectional synaptic plasticity at parallel fiber-Purkinje cell synapses in mice lacking delphilin

Alcohol Impairs Long-Term Depression at the Cerebellar Parallel Fiber–Purkinje Cell Synapse

Acute alcohol consumption causes deficits in motor coordination and gait, suggesting an involvement of cerebellar circuits, which play a role in the fine adjustment of movements and in motor learning. It has previously been shown that ethanol modulates inhibitory transmission in the cerebellum and affects synaptic transmission and plasticity at excitatory climbing fiber (CF) to Purkinje cell synapses. However, it has not been examined thus far how acute ethanol application affects long-term depression (LTD) and long-term potentiation (LTP) at excitatory parallel fiber (PF) to Purkinje cell synapses, which are assumed to mediate forms of cerebellar motor learning. To examine ethanol effects on PF synaptic transmission and plasticity, we performed whole cell patch-clamp recordings from Purkinje cells in rat cerebellar slices. We found that ethanol (50 mM) selectively blocked PF-LTD induction, whereas it did not change the amplitude of excitatory postsynaptic currents at PF synapses. In contrast, ethanol application reduced voltage-gated calcium currents and type 1 metabotropic glutamate receptor (mGluR1)-dependent responses in Purkinje cells, both of which are involved in PF-LTD induction. The selectivity of these effects is emphasized by the observation that ethanol did not impair PF-LTP and that PF-LTP could readily be induced in the presence of the group I mGluR antagonist AIDA or the mGluR1a antagonist LY367385. Taken together, these findings identify calcium currents and mGluR1-dependent signaling pathways as potential ethanol targets and suggest that an ethanol-induced blockade of PF-LTD could contribute to the motor coordination deficits resulting from alcohol consumption.

An Essential Role for PICK1 in NMDA Receptor-Dependent Bidirectional Synaptic Plasticity

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

Steroid-Responsive Chronic Cerebellitis With Positive Glutamate Receptor δ2 Antibody

We report the clinical course of a 4-year-old girl with chronic cerebellitis (onset 2 days after diphtheria-pertussis-tetanus vaccination at 1 year and 7 months old) associated with anti-glutamate receptor delta 2 antibody, who improved dramatically with steroid therapy (methylprednisolone pulse therapy plus oral prednisolone). Recently, it has been reported that the anti-glutamate receptor delta 2 selectively expressed at the post-synaptic site of parallel fiber-Purkinje cell synapses has an important role in cerebellar function in the developing brain. The present case suggests that anti-glutamate receptor delta 2 antibody plays a primary role in an immune-mediated process causing chronic cerebellar symptoms, and the lesion site seems to be localized to the parallel fiber-Purkinje cell synapse. Because the cerebellum is strongly involved in language acquisition as well as motor development, treatment must facilitate time for language learning while reducing the side effects of the corticosteroid therapy.

Postsynaptic GABAB receptor signalling enhances LTD in mouse cerebellar Purkinje cells

Long-term depression (LTD) of excitatory transmission at cerebellar parallel fibre-Purkinje cell synapses is a form of synaptic plasticity crucial for cerebellar motor learning. Around the postsynaptic membrane of these synapses, B-type gamma-aminobutyric acid receptor (GABABR), a Gi/o protein-coupled receptor for the inhibitory transmitter GABA is concentrated and closely associated with type-1 metabotropic glutamate receptors (mGluR1) whose signalling is a key factor for inducing LTD. We found that in cultured Purkinje cells, GABABR activation enhanced LTD of a glutamate-evoked current (LTDglu), increasing the magnitude of depression. It has been reported that parallel fibre-Purkinje cell synapses receive a micromolar level of GABA spilled over from the synaptic terminals of the neighbouring GABAergic interneurons. This level of GABA was able to enhance LTDglu. Our pharmacological analyses revealed that the betagamma subunits but not the alpha subunit of Gi/o protein mediated GABABR-mediated LTDglu enhancement. Gi/o protein activation was sufficient to enhance LTDglu. In this respect, LTDglu enhancement is clearly distinguished from the previously reported GABABR-mediated augmentation of an mGluR1-coupled slow excitatory postsynaptic potential. Baclofen application for only the induction period of LTDglu was sufficient to enhance LTDglu, suggesting that GABABR signalling may modulate mechanisms underlying LTDglu induction. Baclofen augmented mGluR1-coupled Ca2+ release from the intracellular stores in a Gi/o protein-dependent manner. Therefore, GABABR-mediated LTDglu enhancement is likely to result from augmentation of mGluR1 signalling. Furthermore, pharmacological inhibition of GABABR reduced the magnitude of LTD at parallel fibre-Purkinje cell synapses in cerebellar slices. These findings demonstrate a novel mechanism that would facilitate cerebellar motor learning.

Inhibition facilitates depression

The release of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and the subsequent activation of ligand-gated GABAA receptors (GABAAR) and G protein-coupled GABAB receptors (GABABR) is generally thought to, as the name implies, inhibit neurons. On the postsynaptic side, the inhibition produced by GABABR activation is in part mediated by the opening of G protein-coupled inward rectifying potassium (GIRK) channels (Gahwiler & Brown, 1985). Functionally, synaptic activation of GABABR produces a long-lasting (∼400 ms) window of inhibition in a large population of postsynaptic neurons when presynaptic neurons are coherently activated (Scanziani, 2000). A second function of GABABR is the presynaptic inhibition of neurotransmitter release at GABAergic and glutamatergic terminals. As a consequence of this presynaptic action, GABABRs have an influence on the gating of long-term potentiation (LTP) or long-term depression (LTD) in the hippocampus (Davies et al. 1991; Mott & Lewis, 1991; Wagner & Alger, 1995). Here, the spill-over of GABA produced by repetitive stimulation binds to GABABR to inhibit release of GABA during the conditioning and thereby suppresses the gating of LTP or LTD by inhibitory inputs. Yet, outside hippocampus, the role of GABABR activation in the induction of synaptic plasticity remains largely an open question. In the cerebellum, the induction of parallel fibre LTD depends on the conjunctive glutamate-evoked activation of postsynaptic subtype 1 metabotropic glutamate receptors (mGluR1) and depolarization-evoked Ca2+ influx. By activating phospholipase C (PLC) and inducing subsequent release of internal calcium stores, mGluR1 provides an additional calcium influx that will reinforce the activation of protein kinase C (PKC) by depolarization-evoked Ca2+ influx. mGluR1 and GABABR have different first order effectors (Gq/11 and Gi/o protein activation, respectively) yet share many common downstream targets (e.g. IP3R, PLC). An open question is if these common downstream targets endow postsynaptic GABABR with a functional role in the induction of LTD. The exciting work of Kamikubo et al. (2007) in this issue of The Journal of Physiology shows that GABABR activation can facilitate LTD at the parallel fibre–Purkinje cell synapse of the cerebellum, thereby conferring a third major functional role to this receptor on the postsynaptic side (Fig. 1). This facilitation of LTD occurs via the activation of the βγ subunits of the Gi/o protein which in turn activate PLC. This results in an augmentation of the mGluR1 response and an increase in postsynaptic Ca2+ via release from intracellular stores. Quite interestingly, in a study highly reminiscent of this it was shown that the induction of LTP at GABAA synapses in hippocampus is gated by the cooperative activation of GABABRs and group I/II mGluRs via G-protein signalling and intracellular Ca2+ modulation (Patenaude et al. 2003). The similarity of these two studies might indicate that colocalization of GABABR and group I/II mGluRs is sufficient to obtain a cooperative effect of these receptors on synaptic plasticity (Hirono et al. 2001). The idea of cooperation between different G protein-coupled receptors and its role in synaptic plasticity might be far more general than thought until now. Komatsu (1996) showed that the sign and magnitude of synaptic plasticity at cortical inhibitory synapses can be determined by the history of coactivation of GABABR and adrenergic or serotonergic receptors. Recently it was also shown that the magnitude and polarity of spike timing-dependent plasticity is determined not only by the timing between pre- and postsynaptic spikes but also by the recent activity of cholinergic and adrenergic neuromodulators (Seol et al. 2007). Figure 1 Co-activation of mGluR1 and GABABR facilitates induction of cerebellar LTD through the convergence of Gq and Gi/o signaling pathways With the discovery of spike timing-dependent plasticity emerged the idea that coincidence detection might be important for synaptic plasticity in vivo. By showing that coactivation of GABA and glutamate metabotropic receptors facilitates long-term synaptic plasticity in the cerebellum, the recent study from Kamikubo et al. (2007) brings the idea that coincidence of activation of several metabotropic pathways might also be needed in vivo to facilitate synaptic plasticity, or maybe even allow it. By studying the effect of coactivation of different receptor classes sharing common second messenger cascades, new rules for activity-dependent synaptic plasticity could emerge, and will probably reveal that synaptic plasticity relies on networks of proteins far more complex than is usually assumed.

Developmental Changes in Agonist-Induced Retrograde Signaling at Parallel Fiber–Purkinje Cell Synapses: Role of Calcium-Induced Calcium Release

In cerebellar Purkinje cells (PCs), activation of postsynaptic mGluR1 receptors inhibits parallel fiber (PF) to PC synaptic transmission by retrograde signaling. However, results were conflicting with respect to whether endocannabinoids or glutamate (Glu) is the retrograde messenger involved. Experiments in cerebellar slices from 10- to 12-day-old rats and mice confirmed that suppression of PF-excitatory postsynaptic currents (EPSCs) by mGluR1 agonists was entirely blocked by cannabinoid receptor antagonists at this early developmental stage. In contrast, suppression of PF-EPSCs by mGluR1 agonists was only partly blocked by cannabinoid receptor antagonists in 18- to 22-day-old rats, and the remaining suppression was accompanied by an increase in paired-pulse facilitation. This endocannnabinoidindependent suppression of PF-EPSCs was potentiated by the Glu uptake inhibitor D-threo-beta-benzyloxyaspartate (D-TBOA) and blocked by the desensitizing kainate (KA) receptors agonist SYM 2081, by nonsaturating concentrations of 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) [but not by GYKI 52466 hydrochloride (GYKI)] and by dialyzing PCs with guanosine 5'-[beta-thio]diphosphate (GDP-betaS). An endocannnabinoid-independent suppression of PF-EPSCs was also present in nearly mature wild-type mice but was absent in GluR6(-/-) mice. The endocannnabinoid-independent suppression of PF-EPSCs induced by mGluR1 agonists and the KA-dependent component of depolarization-induced suppression of excitation (DSE) were blocked by ryanodine acting at a presynaptic level. We conclude that retrograde release of Glu by PCs participates in mGluR1 agonist-induced suppression of PF-EPSCs at nearly mature PF-PC synapses and that Glu operates through activation of presynaptic KA receptors located on PFs and prolonged release of calcium from presynaptic internal calcium stores.