Induction Of Cerebellar Long-term Depression Research Articles

Inositol 1,4,5-triphosphate receptor is selectively expressed in cerebellum but not cerebellum-like structures of the elasmobranch fish, Leucoraja erinacea

The Inositol 1,4,5-trisphosphate receptor type 1 protein (Ip3r1) performs an essential role for the induction of cerebellar long-term depression. Here, I describe the use of RT-PCR, qPCR, western blotting and immunohistochemistry to assay Ip3r1 gene expression and localize Ip3r1 protein in the hindbrain of the elasmobranch fish, Leucoraja erinacea. Elasmobranchs are representatives of the most basal, yet extant lineage of gnathostomes, or jawed vertebrates. The cerebellum is a synapomorphy for gnathostomes and thus elasmobranch cerebellar physiology may serve as a proxy for the ancestral state of other jawed vertebrates. LeIp3r1 is selectively expressed in the cerebellum of the little skate and the resultant protein is localized to Purkinje cells. If Ip3r1 performs the same functions in the skate cerebellum as in the mammalian cerebellum, then parallel fiber-Purkinje cell long-term depression through Ip3r1 mediated intracellular calcium regulation may be a conserved feature of cerebellar physiology. Cerebellum and surrounding hindbrain regions termed cerebellum-like structures share a common developmental genetic toolkit. LeIp3r1 expression was lowly detected in cerebellum-like structures indicating that although generatively homologous, the cerebellum and cerebellum-like structures do not share a complete overlap of common expression. Because of the little skate's important phylogenetic placement, performing molecular methodologies to assay targeted gene expression and determine protein localization in the hindbrain can be valuable for our understanding of cerebellar evolution and comparative neural development.

A Model of Induction of Cerebellar Long-Term Depression Including RKIP Inactivation of Raf and MEK.

We report an updated stochastic model of cerebellar Long Term Depression (LTD) with improved realism. Firstly, we verify experimentally that dissociation of Raf kinase inhibitor protein (RKIP) from Mitogen-activated protein kinase kinase (MEK) is required for cerebellar LTD and add this interaction to an earlier published model, along with the known requirement of dissociation of RKIP from Raf kinase. We update Ca2+ dynamics as a constant-rate influx, which captures experimental input profiles accurately. We improve α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptor interactions by adding phosphorylation and dephosphorylation of AMPA receptors when bound to glutamate receptor interacting protein (GRIP). The updated model is tuned to reproduce experimental Ca2+ peak vs. LTD amplitude curves at four different Ca2+ pulse durations as closely as possible. We find that the updated model is generally more robust with these changes, yet we still observe some sensitivity of LTD induction to copy number of the key signaling molecule Protein kinase C (PKC). We predict natural variability in this number by stochastic diffusion may influence the probabilistic LTD response to Ca2+ input in Purkinje cell spines and propose this as an extra source of stochasticity that may be important also in other signaling systems.

Functional and Physical Interaction of Diacylglycerol Kinase ζ with Protein Kinase Cα Is Required for Cerebellar Long-Term Depression.

Many studies have identified signaling molecules that mediate long-term synaptic plasticity. In the basal state, the activities and concentrations of these signaling molecules must be maintained at low levels, yet be ready to be boosted, so that synapses can undergo synaptic plasticity only when they are stimulated. However, the mechanisms involved in creating such conditions are not well understood. Here, we show that diacylglycerol kinase ζ (DGKζ) creates optimal conditions for the induction of cerebellar long-term depression (LTD). DGKζ works by regulating localization and activity of protein kinase Cα (PKCα), an important mediator of LTD, so that PKCα effectively responds to the stimulation that triggers LTD.

Polyglutamine-expanded ataxin-3 impairs long-term depression in Purkinje neurons of SCA3 transgenic mouse by inhibiting HAT and impairing histone acetylation

Our previous study using a transgenic mouse model of spinocerebellar ataxia type 3 (SCA3) reported that disease-causing ataxin-3-Q79 caused cerebellar malfunction by inducing transcriptional downregulation. Long-term depression (LTD) of parallel fiber-Purkinje neuron glutamatergic transmission is believed to be a cellular mechanism for motor learning and motor coordination in the cerebellum. Downregulated mRNA expression of calcineurin B, IP3-R1, myosin Va and PLC β4, which are required for the induction of cerebellar LTD, led to an impairment of LTD induction in Purkinje neurons of SCA3 transgenic mouse. Our study suggested that ataxin-3-Q79 caused hypoacetylation of cerebellar histone H3 or H4 by inhibiting the activity of histone acetyltransferase (HAT) without affecting the activity of histone deacetylase (HDAC). Consistent with the hypothesis that hypoacetylated H3 or H4 histone associated with promoter regions of downregulated genes is the molecular mechanism underlying ataxin-3-Q79-induced transcriptional repression, chromatin immunoprecipitation-quantitative real-time PCR analysis showed hypoacetylation of H3 or H4 histone associated with the proximal promoter of downregulated calcineurin B, IP3-R1, myosin Va or PLC β4 gene in the cerebellum of SCA3 mouse. HDAC inhibitor sodium butyrate reversed ataxin-3-Q79-induced hypoacetylation of histone H3 or H4 associated with the proximal promoter of calcineurin B, IP3-R1, myosin Va or PLC β4 gene. Sodium butyrate also prevented ataxin-3-Q79-induced impairment of LTD induction in Purkinje neurons of SCA3 mice. Our results suggest that polyglutamine-expanded ataxin-3-Q79 impairs HAT activity, leading to histone hypoacetylation, downregulated expression of cerebellar genes required for LTD induction and impaired induction of cerebellar LTD in the SCA3 transgenic mouse.

Initial state of single spines affects probability of induction of cerebellar long term depression in a stochastic model

Cerebellar long-term depression (LTD) is a type of synaptic plasticity which shows a robust and persistent decrease in the synaptic transmission between parallel fibers (PF) and Purkinje cells (PC), expressed as a reduction in the number of synaptic AMPA receptors. Our lab recently developed a stochastic model of the LTD signaling network, including a PKC-ERK-cPLA2 positive feedback loop and mechanisms of AMPAR trafficking, and tuned the model to replicate calcium uncaging experiments [1]. Probabilistically, signaling activity switches between two discrete stable states (LTD or non-LTD). In single spines, the probability of LTD occurrence is only modulated by the concentration and duration of the signal used to trigger it, and inputs with the same magnitude give rise to one of two responses. The detailed kinetic model of the signaling and trafficking network involved in the induction of cerebellar LTD was developed based on extensive experimental data. The model was solved stochastically and deterministically using STEPS (http://steps.sourceforge.net/), a well validated simulator that implements the Stochastic Simulation Algorithm (SSA)[2]. The reasons for the bistable behavior in single spines is not fully understood. Molecular memory of the compartment, i.e. initial state of molecules before calcium injection, may play a role in altering the probability of a spine to undergo LTD induction or not. We address the possible effects of this initial state on the observed bistable behavior quantitatively. Preliminary analysis of these initial conditions for several simulations lead to their categorization on the basis of LTD probability observed for a particular calcium concentration and pulse duration (for simplicity we named them as High LTD likelihood, Normal LTD likelihood and Low LTD likelihood). We then confirmed these categories for different calcium conditions. The probability of LTD induction has a dependence on calcium pulse duration well-described by the Hill equation. The High and Normal LTD likelihood cases showed similar characteristics, though the Low LTD likelihood category showed different Hill parameters and also didn't saturate at 100 percent. Observations when varying the time before calcium input showed that the High and Normal LTD likelihood states exist only on the order of minutes and dissipate afterwards. In depth understanding of these different initial states may give better insights into the underlying molecular mechanisms causing bistable behavior. This is an important phenomena, as it is also observed in other forms of long-term synaptic plasticity and in many other biological processes.

TRPC3 Channel Underlies Cerebellar Long-Term Depression

Cerebellar long-term depression (LTD) is induced by repetitive pairing of both synaptic inputs provided by climbing fibers (CFs) and parallel fibers (PFs), especially when CF stimulation followed by burst of PFs. Metabotropic glutamate receptor type 1 (mGluR1)-dependent signaling in Purkinje cells is critically involved in the induction of cerebellar LTD. Signaling pathway of mGluR1 has two limbs: one is IP3 receptor-mediated Ca release from intracellular Ca store and the other is activation of transient receptor potential canonical (TRPC) channels. Here, we hypothesized that TRPC3, which is reported to be responsible for mGluR1-evoked slow currents, mediates the induction of the cerebellar LTD. Purkinje cells were loaded with antibodies which act against a cytoplasmic epitope of TRPC3 channels, acting as a specific antagonist of TRPC3. About 30min after achieving a whole-cell configuration, mGluR1-evoked slow currents were significantly reduced; a possible cause of this time delay might be the diffusion time course from patch pipette to the dendrite site of mGluR1 activation. Taking this delay into consideration, we next attempted to induce cerebellar LTD by pairing PFs and CF. It was found that cerebellar LTD was hindered in the presence of TRPC3 antibodies. Taken together, our data suggest that TRPC3 activation may be essential for the induction of LTD in cerebellar Purkinje cells.

A common framework of signal processing in the induction of cerebellar LTD and cortical STDP

Cerebellar long-term depression (LTD) and cortical spike-timing-dependent synaptic plasticity (STDP) are two well-known and well-characterized types of synaptic plasticity. Induction of both types of synaptic plasticity depends on the spike timing, pairing frequency, and pairing numbers of two different sources of spiking. This implies that the induction of synaptic plasticity may share common frameworks in terms of signal processing regardless of the different signaling pathways involved in the two types of synaptic plasticity. Here we propose that both types share common frameworks of signal processing for spike-timing, pairing-frequency, and pairing-numbers detection. We developed system models of both types of synaptic plasticity and analyzed signal processing in the induction of synaptic plasticity. We found that both systems have upstream subsystems for spike-timing detection and downstream subsystems for pairing-frequency and pairing-numbers detection. The upstream systems used multiplication of signals from the feedback filters and nonlinear functions for spike-timing detection. The downstream subsystems used temporal filters with longer time constants for pairing-frequency detection and nonlinear switch-like functions for pairing-numbers detection, indicating that the downstream subsystems serve as a leaky integrate-and-fire system. Thus, our findings suggest that a common conceptual framework for the induction of synaptic plasticity exists despite the differences in molecular species and pathways.

Transient Receptor Potential Canonical Channels Regulate the Induction of Cerebellar Long-Term Depression

In the cerebellum, synaptic strength at the synapses between parallel fibers and Purkinje cells is best known to be modulated via metabotropic glutamate receptor 1 (mGluR1)-dependent cerebellar long-term depression (LTD). An increase in intracellular calcium levels plays an important role in inducing mGluR1-dependent cerebellar LTD. Downstream of mGluR1, there are two major sources of calcium: transient receptor potential canonical (TRPC) channels and inositol trisphosphate receptors (IP(3)R). IP(3)R triggers a calcium release from the intracellular calcium store. Here, we show that TRPC channels mediate mGluR1-evoked slow currents to regulate cerebellar LTD in Sprague Dawley rats. We found that the inhibition of TRPC channels blocks the induction of cerebellar LTD. Moreover, we show that processes known to underlie cerebellar LTD induction, such as increases in intracellular calcium concentration, the activation of protein kinase C, and the internalization of GluR2, are also hindered by blocking TRPC. These results suggest that the mGluR1-evoked activation of TRPC channels is required for the induction of cerebellar LTD.

A Stochastic Signaling Network Mediates the Probabilistic Induction of Cerebellar Long-Term Depression

Many cellular processes involve a small number of molecules and undergo stochastic fluctuations in their levels of activity. Cerebellar long-term depression (LTD) is a form of synaptic plasticity expressed as a reduction in the number of synaptic AMPA receptors (AMPARs) in Purkinje cells. We developed a stochastic model of the LTD signaling network, including a PKC-ERK-cPLA(2) positive feedback loop and mechanisms of AMPAR trafficking, and tuned the model to replicate calcium uncaging experiments. The signaling network activity in single synapses switches between two discrete stable states (LTD and non-LTD) in a probabilistic manner. The stochasticity of the signaling network causes threshold dithering and allows at the macroscopic level for many different and stable mean magnitudes of depression. The probability of LTD occurrence in a single spine is only modulated by the concentration and duration of the signal used to trigger it, and inputs with the same magnitude can give rise to two different responses; there is no threshold for the input signal. The stochasticity is intrinsic to the signaling network and not mostly dependent on noise in the calcium input signal, as has been suggested previously. The activities of the ultrasensitive ERK and of cPLA(2) undergo strong stochastic fluctuations. Conversely, PKC, which acts as a noise filter, is more constantly activated. Systematic variation of the biochemical population size demonstrates that threshold dithering and the absence of spontaneous LTD depend critically on the number of molecules in a spine, indicating constraints on spine size in Purkinje cells.

Phosphoinositide Signaling Regulates the Surface Localization of the δ2 Ionotropic Glutamate Receptor

The δ2 glutamate receptor (GluD2) is a member of the ionotropic glutamate receptor family. Since GluD2 remains an orphan receptor, it has not been shown to conduct ions. It is highly expressed in the parallel fiber-Purkinje cell (PF-PC) synapse and its role in cerebellar physiology is increasingly appreciated. GluD2 has been linked to the induction of cerebellar long-term depression (LTD) and presynaptic terminal differentiation. A single point mutation in the second transmembrane domain (A654T), named Lurcher (GluD2Lc), confers constitutive activity to the receptor.We have used the Lurcher mutant as a model to examine whether phosphoinositides regulate GluD2. We show that decreasing PIP2 levels in the membrane, through Gq-coupled receptor activation or pretreatment with a PI4K inhibitor, potentiates GluD2Lc currents. Conversely, increasing PIP2 levels by co-expressing PIP5K leads to decreased GluD2Lc currents. On the other hand, utilizing PI3K inhibitors wortmannin or LY294002, and presumably decreasing PIP3 levels, reduces GluD2Lc currents, while co-expression of PI3K leads to potentiation of GluD2Lc currents.A chemiluminescence-based assay that quantifies surface localization of the GluD2 and GluD2Lc receptors, showed that manipulations of the membrane phosphoinositide levels evoke changes in the cell surface localization of both wild-type and mutant receptors. These changes in surface localization of the receptor correspond to the effects we have observed by monitoring GluD2Lc currents, suggesting that current measurements from this mutant receptor serve as a good reporter for the localization of the wild-type δ2 receptor. These results are consistent with the interpretation that increased PIP2 levels decrease, while increased PIP3 levels increase localization of the receptor at the cell surface. Signals that affect the levels of these phosphoinositides simultaneously are likely to regulate the surface localization of GluD2 based on the net change of the two opposing effects.

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.

Functional roles of neuropeptides in cerebellar circuits

Whereas the cerebellum contains 22 different types of neuropeptides as presently known, their expression is generally weak and diffusely dispersed in cerebellar tissues, which often makes their functional significance doubtful. Nevertheless, our knowledge about certain neuropeptides has advanced to the extent that we can figure out their unique functional roles in cerebellar circuits. Throughout the cerebellum, CRF is contained in climbing fibers and its spontaneous release is required for the induction of cerebellar long-term depression (LTD), a cellular mechanism of motor learning. Corticotropin-releasing factor (CRF) is also expressed in the paraventricular nucleus–pituitary system and amygdala–lower brainstem system, both of which are involved in coping responses to stress. In view that motor learning requires stressful efforts for correcting errors in repeated trials, CRF in climbing fibers may imply that the olivocerebellar system is part of a large CRF-operated functional system that acts to cope with various stressors. Orexin, on the other hand, is contained in beaded fibers, which, originating from the hypothalamus, project to various brainstem nuclei and also to the cerebellum, exclusively the flocculus. Currently available evidence suggests that, in fight-or-flight situations, orexinergic neurons switch the state of cardiovascular control systems including the flocculus to secure blood supply to working muscles. Considerable knowledge has also been accumulated about angiotensin II, galanin, and cerebellin, but there is still a gap in defining their unique functional roles in cerebellar circuits.

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.

The δ2 ‘ionotropic’ glutamate receptor functions as a non‐ionotropic receptor to control cerebellar synaptic plasticity

The delta2 glutamate receptor (GluRdelta2) belongs to the ionotropic glutamate receptor (iGluR) family and plays a crucial role in the induction of cerebellar long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Nevertheless, the mechanisms by which GluRdelta2 regulates cerebellar LTD have remained elusive. Because a mutation occurring in lurcher mice causes continuous GluRdelta2 channel activity that can be abolished by 1-naphtylacetylspermine (NASP), a channel blocker for Ca(2+)-permeable iGluRs, GluRdelta2 is thought to function as an ion channel. Here, we introduced a mutant GluRdelta2 transgene, in which the putative channel pore was disrupted, into GluRdelta2-null Purkinje cells using a virus vector. Surprisingly and similar to the effect of the wild-type GluRdelta2 transgene, the mutant GluRdelta2 completely rescued the abrogated LTD in GluRdelta2-null mice. Furthermore, NASP did not block LTD induction in wild-type cerebellar slices. These results indicate that GluRdelta2, a member of the iGluR family, does not serve as a channel in the regulation of LTD induction.

Timing dependence of the induction of cerebellar LTD

Long-term depression (LTD) of the granule cell to Purkinje cell synapse is thought to contribute to motor learning. According to the Marr/Albus/Ito model, sensory inputs drive granule cells to fire, thereby exciting Purkinje cells and influencing motor output. Inappropriate motor output causes neurons in the inferior olive to fire and activate Purkinje cells via the powerful climbing fiber (CF) synapse. CF activity is an error signal and the association of CF and granule cell parallel fiber (PF) activity results in LTD at coactivated PF synapses. Here we examine the timing dependence of LTD by using an induction protocol consisting of a single CF activation paired with a PF burst, with the relative timing of CF and PF activation systematically varied. LTD was most prominent when PF activation occurred before CF activation. A plot of LTD magnitude as a function of PF and CF timing was well approximated by a fit in which LTD peaked for PF activity ∼80ms before CF activation and the half width was ∼300ms. This indicates that the timing dependence of LTD is well suited to allow a CF to depress preceding PF inputs that generated inappropriate motor outputs. We also find that LTD induction and endocannabinoid release have a similar dependence on PF and CF timing. This suggests that the properties of endocannabinoid release may underlie the timing dependence of some forms of motor learning.

Induction of cerebellar long-term depression requires activation of calcineurin in Purkinje cells

Cerebellar long-term depression (LTD) is an activity-dependent depression of synaptic transmission from parallel fibers to Purkinje cells underlying certain forms of motor learning. LTD is induced by the conjunctive stimulation of parallel fibers and climbing fibers, both of which supply excitatory inputs to Purkinje cells. The conjunctive stimulation induces a large increase in intracellular Ca 2+ concentration ([Ca 2+] i) in Purkinje cells. Although the increase in [Ca 2+] i is essential for LTD induction, the downstream signal transduction mechanism remains elusive. In this study, we show that LTD induction requires the activation of the Ca 2+/calmodulin-dependent protein phosphatase 2B calcineurin. In acute cerebellar slices of mice, the LTD amplitude was significantly reduced in the presence of calcineurin inhibitors (cyclosporin A or FK506), whereas the basic electrophysiological properties of the parallel fiber-Purkinje cell synaptic transmission remained constant. Furthermore, a calcineurin autoinhibitory peptide perfused into Purkinje cells completely blocked LTD induction. On the other hand, microcystin LR, an inhibitor of protein phosphatase 1 and 2A, did not affect the induction of LTD. These results indicate that calcineurin activation is essential for LTD induction downstream of the conjunctive-stimulation-induced Ca 2+ signal in Purkinje cells.

Involvement of Na +–Ca 2+ exchanger on metabotropic glutamate receptor 1-mediated [Ca 2+] i transients in rat cerebellar Purkinje neurons

Cerebellar Purkinje neurons have intracellular regulatory systems including Ca 2+-binding proteins, intracellular Ca 2+ stores, Ca 2+-ATPase and Na +–Ca 2+ exchanger (NCX) that keep intracellular Ca 2+ concentration ([Ca 2+] i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca 2+] i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca 2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca 2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca 2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.

Selective Engagement of Plasticity Mechanisms for Motor Memory Storage

The number and diversity of plasticity mechanisms in the brain raises a central question: does a neural circuit store all memories by stereotyped application of the available plasticity mechanisms, or can subsets of these mechanisms be selectively engaged for specific memories? The uniform architecture of the cerebellum has inspired the idea that plasticity mechanisms like cerebellar long-term depression (LTD) contribute universally to memory storage. To test this idea, we investigated a set of closely related, cerebellum-dependent motor memories. In mutant mice lacking Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), the maintenance of cerebellar LTD is abolished. Although memory for an increase in the gain of the vestibulo-ocular reflex (VOR) induced with high-frequency stimuli was impaired in these mice, memories for decreases in VOR gain and increases in gain induced with low-frequency stimuli were intact. Thus, a particular plasticity mechanism need not support all cerebellum-dependent memories, but can be engaged selectively according to the parameters of training.

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.

An NMDA Receptor/Nitric Oxide Cascade Is Involved in Cerebellar LTD But Is Not Localized to the Parallel Fiber Terminal

Long-term depression (LTD) of the parallel fiber-Purkinje cell synapse in the cerebellum is a cellular model system that has been suggested to underlie certain forms of motor learning. Induction of cerebellar LTD requires a postsynaptic kinase limb involving activation of mGluR1, protein kinase Calpha (PKCalpha), and phosphorylation of ser-880 on the AMPA receptor subunit GluR2. Several lines of evidence have also implicated a complementary phosphatase limb in which N-methyl-d-aspartate (NMDA) receptor-mediated Ca(2+) influx activates neuronal nitric oxide synthase (nNOS), the ultimate consequences of which are mediated by nitric oxide (NO), cGMP, and inhibition of postsynaptic protein phosphatases. However, the cellular localization of an NMDA/NO cascade has been complicated by the fact that neither functional NMDA receptors nor nNOS are expressed in Purkinje cells. This has lead to a proposal in which NMDA receptors activate nNOS in parallel fibers. Here, we confirm that pharmacological blockade of NMDA receptor or NO signaling blocks induction of LTD. However, no evidence was found for functional NMDA receptors in parallel fiber terminals: blockade of NMDA receptors did not alter either presynaptic Ca(2+) transients or the frequency of miniature excitatory postsynaptic currents. NMDA receptor blockade did abolish a slow depolarization evoked by burst stimulation of parallel fiber-stellate cell synapses. The application of NMDA evoked a Ca(2+) transient in stellate cell terminals but not in parallel fiber terminals. These results are consistent with the hypothesis that an NMDA receptor/NO cascade involved in cerebellar LTD is localized to interneurons rather than parallel fibers.