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Simple and regular anatomical structure is a hallmark of the cerebellar cortex. Parasagittally arrayed alternate expression of aldolase C/zebrin II in Purkinje cells (PCs) has been extensively studied, but surprisingly little is known about its functional significance. Here we found a precise structure-function relationship between aldolase C expression and synchrony of PC complex spike activities that reflect climbing fiber inputs to PCs. We performed two-photon calcium imaging in transgenic mice in which aldolase C compartments can be visualized in vivo, and identified highly synchronous complex spike activities among aldolase C-positive or aldolase C-negative PCs, but not across these populations. The boundary of aldolase C compartments corresponded to that of complex spike synchrony at single-cell resolution. Sensory stimulation evoked aldolase C compartment-specific complex spike responses and synchrony. This result further revealed the structure-function segregation. In awake animals, complex spike synchrony both within and between PC populations across the aldolase C boundary were enhanced in response to sensory stimuli, in a way that two functionally distinct PC ensembles are coactivated. These results suggest thatPCpopulations characterized by aldolaseCexpression precisely represent distinct functional units of the cerebellar cortex, and these functional units can cooperate to process sensory information in awake animals. [ABSTRACT FROM AUTHOR]

Endocannabinoids are known to mediate retrograde suppression of synaptic transmission, modulate synaptic plasticity, and influence learning and memory. The 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase α (DGLα) is regarded as the major endo-cannabinoid that causes retrograde synaptic suppression. To determine how 2-AG signaling influences learning and memory, we subjected DGLα knock-out mice to two learning tasks. We tested the mice using habituation and odor-guided transverse patterning tasks that are known to involve the dentate gyrus and the CA1, respectively, of the hippocampus. We found that DGLα knock-out mice showed significantly faster habituation to an odor and a new environment than wild-type littermates with normal performance in the transverse patterning task. In freely moving animals, long-term potentiation (LTP) induced by theta burst stimulation was significantly larger at perforant path- granule cell synapses in the dentate gyrus of DGLα knock-out mice. Importantly, prior induction of synaptic potentiation at this synapse caused a significant retardation of habituation in DGLα knock-out but not in wild-type littermates. The excitability of granule cells became higher in DGLα knock-out mice after they generated action potentials. Since no differences were found in intrinsic membrane properties and responses to odor stimuli in granule cells, the elevated excitability is considered to result from enhanced activity of an excitatory recurrent network composed of granule cells and mossy cells. These results suggest that retrograde 2-AG signaling negatively regulates habituation by suppressing excitatory recurrent network activity and reducing LTP in the dentate gyrus. [ABSTRACT FROM AUTHOR]

Functional clustering of neurons is frequently observed in the motor cortex. However, it is unknown if, when, and how fine-scale (<100 μm) functional clusters form relative to voluntary forelimb movements. In addition, the implications of clustering remain unclear. To address these issues, we conducted two-photon calcium imaging of mouse layer 2/3 motor cortex during a self-initiated lever-pull task. In the imaging session after 8-9 days of training, head-restrained mice had to pull a lever for ~600 ms to receive a water drop, and then had to wait for >3 s to pull it again. We found two types of task-related cells in the mice: cells whose peak activities occurred during lever pulls (pull cells) and cells whose peak activities occurred after the end of lever pulls. The activity of pull cells was strongly associated with lever-pull duration. In ~40% of imaged fields, functional clusterings were temporally detected during the lever pulls. Spatially, there were ~70μm-scale clusters that consisted of more than four pull cells in ~50% of the fields. Ensemble and individual activities of pull cells within the cluster more accurately predicted lever movement trajectories than activities of pull cells outside the cluster. This was likely because clustered pull cells were more often active in the individual trials than pull cells outside the cluster. This higher fidelity of activity was related to higher trial-to-trial correlations of activities of pairs within the cluster. We propose that strong recurrent network clusters may represent the execution of voluntary movements. [ABSTRACT FROM AUTHOR]

We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary expiant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures. PCs were initially reinnervated by multiple CFs with similar strengths. Surplus CFs were eliminated subsequently, and the remaining CFs became stronger. These changes are similar to those occurring in developing cerebellum in vivo. Importantly, the changes in CF innervations in cocultures involved the same molecules required for CF synapse elimination in vivo, including NMDA receptor, type 1 metabotropic glutamate receptor and glutamate receptor &dgr;2 (GluR&dgr;2). We demonstrate that gain- and loss-of-function analyses can be efficiently performed by lentiviral-mediated overexpression and RNAi-induced knockdown of GluR&dgr;2. Using this approach, we identified neuroligin-2 as a novel molecule that promotes CF synapse elimination in postsynaptic PCs. Thus, our coculture preparation will greatly facilitate the elucidation of molecular mechanisms of synapse elimination. [ABSTRACT FROM AUTHOR]

In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs wasmost developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABAA receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABAA receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals. [ABSTRACT FROM AUTHOR]

2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase_ (DGLα) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGLα-knock-out mice. Immunohistochemistry revealed that DGLα was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB1 receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MCterminals making synapses with other GCspines in the neighborhood. MGL-expressing elements, however, poorly covered GCspines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation. [ABSTRACT FROM AUTHOR]

Mutations of the myosin Va gene cause the neurological diseases Griscelli syndrome type 1 and Elejalde syndrome in humans and dilute phenotypes in rodents. To understand the pathophysiological mechanisms underlying the neurological disorders in myosin Va diseases, we conducted an integrated analysis at the molecular, cellular, electrophysiological, and behavioral levels using the dilute-neurological (d-n) mouse mutant. These mice manifest an ataxic gait and clonic seizures during postnatal development, but the neurological disorders are ameliorated in adulthood. We found that smooth endoplasmic reticulum (SER) rarely extended into the dendritic spines of Purkinje cells (PCs) of young d-n mice, and there were few, if any, IP3 receptors. Moreover, long-term depression (LTD) at parallel fiber-PC synapses was abolished, consistent with our previous observations in juvenile lethal dilute mutants. Young d-n mice exhibited severe impairment of cerebellum-dependent motor learning. In contrast, adult d-n mice showed restoration of motor learning and LTD, and these neurological changes were associated with accumulation of SER and IP3 receptors in some PC spines and the expression of myosin Va proteins in the PCs. RNA interference-mediated repression of myosin Va caused a reduction in the number of IP3 receptor-positive spines in cultured PCs. These findings indicate that myosin Va function is critical for subsequent processes in localization of SER and IP3 receptors in PC spines, LTD, and motor learning. Interestingly, d-n mice had defects of motor coordination from young to adult ages, suggesting that the role of myosin Va in PC spines is not sufficient for motor coordination. [ABSTRACT FROM AUTHOR]

Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB1 receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid 2-arachidonoylglycerol (2-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca2+ elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB1 receptors and suppresses transmitter release at hippocampal inhibitory synapses. [ABSTRACT FROM AUTHOR]

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s. [ABSTRACT FROM AUTHOR]

Endogenous cannabinoids (endocannabinoids) mediate retrograde signals for short- and long-term suppression of transmitter release at synapses of striatal medium spiny (MS) neurons. An endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is synthesized from diacylglycerol (DAG) after membrane depolarization and Gq-coupled receptor activation. To understand 2-AG-mediated retrograde signaling in the striatum, we determined precise subcellular distributions of the synthetic enzyme of 2-AG, DAG lipase-α (DAGLα), and its upstream metabotropic glutamate receptor 5 (mGluR5) and muscarinic acetylcholine receptor 1 (M1). DAGLα, mGluR5, and M1 were all richly distributed on the somatodendritic surface of MS neurons, but their subcellular distributions were different. Although mGluR5 and DAGLα levels were highest in spines and accumulated in the perisynaptic region, M1 level was lowest in spines and was rather excluded from the mGluR5-rich perisynaptic region. These subcellular arrangements suggest that mGluR5 and M1 might differentially affect endocannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) in MS neurons. Indeed, mGluR5 activation enhanced both DSI and DSE, whereas M1 activation enhanced DSI only. Importantly, DSI, DSE, and receptor-driven endocannabinoid-mediated suppression were all abolished by the DAG lipase inhibitor tetrahydrolipstatin, indicating 2-AG as the major endocannabinoid mediating retrograde suppression at excitatory and inhibitory synapses of MS neurons. Accordingly, CB1 cannabinoid receptor, the main target of 2-AG, was present at high levels on GABAergic axon terminals of MS neurons and parvalbumin-positive interneurons and at low levels on excitatory corticostriatal afferents. Thus, endocannabinoid signaling molecules are arranged to modulate the excitability of the MS neuron effectively depending on cortical activity and cholinergic tone as measured by mGluR5 and M1 receptors, respectively. [ABSTRACT FROM AUTHOR]

Endocannabinoids function as retrograde messengers and modulate synaptic transmission through presynaptic cannabinoid CB1 receptors. The magnitude and time course of endocannabinoid signaling are thought to depend on the balance between the production and degradation of endocannabinoids. The major endocannabinoid 2-arachidonoylglycerol (2-AG) is hydrolyzed by monoacylglycerol lipase (MGL), which is shown to be localized at axon terminals. In the present study, we investigated how MGL regulates endocannabinoid signaling and influences synaptic transmission in the hippocampus. We found that MGL inhibitors, methyl arachidonoyl fluorophosphonate and arachidonoyl trifluoromethylketone, caused a gradual suppression of cannabinoid-sensitive IPSCs in cultured hippocampal neurons. This suppression was reversed by blocking CB1 receptors and was attenuated by inhibiting 2-AG synthesis, indicating that MGL scavenges constitutively released 2-AG. We also found that the MGL inhibitors significantly prolonged the suppression of both IPSCs and EPSCs induced by exogenous 2-AG and depolarization-induced suppression of inhibition/excitation, a phenomenon known to be mediated by retrograde endocannabinoid signaling. In contrast, inhibitors of other endocannabinoid hydrolyzing enzymes, fatty acid amide hydrolase and cyclooxygenase-2, had no effect on the 2-AG-induced IPSC suppression. These results strongly suggest that presynaptic MGL not only hydrolyzes 2-AG released from activated postsynaptic neurons but also contributes to degradation of constitutively produced 2-AG and prevention of its accumulation around presynaptic terminals. Thus, the MGL activity determines basal endocannabinoid tone and terminates retrograde endocannabinoid signaling in the hippocampus. [ABSTRACT FROM AUTHOR]

Tonically active cholinergic interneurons in the striatum modulate activities of striatal outputs from medium spiny (MS) neurons and significantly influence overall functions of the basal ganglia. Cellular mechanisms of this modulation are not fully understood. Here we show that ambient acetylcholine (ACh) derived from tonically active cholinergic interneurons constitutively upregulates depolarization-induced release of endocannabinoids from MS neurons. The released endocannabinoids cause transient suppression of inhibitory synaptic inputs to MS neurons through acting retrogradely onto presynaptic CB1 cannabinoid receptors. The effects were mediated by postsynaptic M1 subtype of muscarinic ACh receptors, because the action of a muscarinic agonist to release endocannabinoids and the enhancement of depolarization-induced endocannabinoid release by ambient ACh were both deficient in M1 knock-out mice and were blocked by postsynaptic infusion of guanosine-5′-0-(2-thiodiphosphate). Suppression of spontaneous firings of cholinergic interneurons by inhibiting Ih current reduced the depolarization-induced release of endocannabinoids. Conversely, elevation of ambient ACh concentration by inhibiting choline esterase significantly enhanced the endocannabinoid release. Paired recording from a cholinergic interneuron and an MS neuron revealed that the activity of single cholinergic neuron could influence endocannabinoid-mediated signaling in neighboring MS neurons. These results clearly indicate that striatal endocannabinoid-mediated modulation is under the control of cholinergic interneuron activity. By immunofluorescent and immunoelectron microscopic examinations, we demonstrated that M1 receptor was densely distributed in perikarya and dendrites of dopamine D1 or D2 receptor-positive MS neurons. Thus, we have disclosed a novel mechanism by which the muscarinic system regulates striatal output and may contribute to motor control. [ABSTRACT FROM AUTHOR]

Cannabinoids exert their psychomotor actions through the CB1 cannabinoid receptor in the brain. Genetic deletion of CB1 in mice causes various symptoms, including changes in locomotor activity, increased ring catalepsy, supraspinal hypoalgesia, and impaired memory extinction. Although the cerebellar cortex contains the highest level of CB1, severe cerebellum-related functional deficits have not been reported in CB1 knock-out mice. To clarify the roles of CB1 in cerebellar function, we subjected CB1 knock-out mice to a delay version of classical eyeblink conditioning. This paradigm is a test for cerebellum-dependent discrete motor learning, in which conditioned stimulus (CS) (352m stone) and unconditioned stimulus (US) (100ms periorbital electrical shock) are coterminated. We found that delay eyeblink conditioning performance was severely impaired in CB1 knock-out mice. In contrast, they exhibited normal performance in a trace version of eyeblink conditioning with 500 ms stimulus-free interval intervened between the CS offset and the US onset. This paradigm is a test for hippocampus-dependent associative learning. Sensitivity of CB1 knock-out mice to CS or US was normal, suggesting that impaired delay eyeblink conditioning is attributable to defects in association of responses to CS and US. We also found that intraperitoneal injection of the CB1 antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide] to wild-type mice caused severe impairment in acquisition but not extinction of delay eyeblink conditioning. SR141716A treatment had no effect on trace eyeblink conditioning with a 500 or 750ms trace interval. These results indicate that endogenous cannabinoid signaling through CB1 is essential for cerebellum-dependent discrete motor learning, especially for its acquisition. [ABSTRACT FROM AUTHOR]

2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-α (DAGLα), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLα was predominantly expressed in Purkinje cells. DAGLα was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLα was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLα was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLα was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLα was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLα is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types. [ABSTRACT FROM AUTHOR]

Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocannabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, "CB3," and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s) of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specific antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole- 3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum. [ABSTRACT FROM AUTHOR]

Classical conditioning of the eyeblink reflex is a simple form of associative learning for motor responses. To examine the involvement of hippocampal CA3 NMDA receptors (NRs) in nonspatial associative memory, mice lacking an NR1 subunit selectively in adult CA3 pyramidal cells [CA3-NR1 knock-out (KO) mice] were subjected to eyeblink conditioning paradigms. Mice received paired presentations of an auditory conditioned stimulus (CS) and a periorbital shock unconditioned stimulus (US). With repeated presentation of the CS followed by the US, wild-type mice learned to blink in anticipation of the US before its onset. We first confirmed that wild-type mice require an intact hippocampus in the trace version of eyeblink conditioning in which the CS and US do not overlap, creating a stimulus-free time gap of 500 ms. Under the same condition, CA3-NR1 KO mice successfully acquired conditioned responses (CRs) during the 10 d acquisition sessions, whereas the extinction of CRs was impaired on the first day of extinction sessions. Importantly, CA3-NR1 KO mice were impaired in the formation of an adaptively timed CR during the first five trials in the daily acquisition sessions. The aberrantly timed CR was also observed in the extinction sessions in accordance with the impaired extinction of CRs. These results indicate that CA3-NR1 KO mice are unable to rapidly retrieve adaptive CR timing, suggesting that CA3 NRs play a crucial role in the memory of adaptive CR timing in trace conditioning. [ABSTRACT FROM AUTHOR]

Activation of cannabinoid receptors suppresses neurotransmitter release in various brain regions. In cerebellar Purkinje cells (PCs), cannabinoid agonists suppress both EPSC and IPSC evoked by stimulating the corresponding inputs. However, cannabinoid agonists suppress miniature IPSC (mIPSC) but not miniature EPSC (mEPSC) at normal external Ca2+ concentration ([Ca2+]o). Therefore, cannabinoid agonists are thought to suppress release machinery for IPSCs but not that for EPSCs. Here we investigated the possible cause of this difference and found that cannabinoid agonists selectively suppressed Ca2+-enhanced miniature events. A cannabinoid agonist, WIN55,212-2 (5 µM), did not affect mEPSC frequency with 2 mM extracellular Ca2+ (Ca2+o). However, WIN55,212-2 became effective when mEPSC frequency was enhanced by elevation of presynaptic Ca2+level by perfusion with 5mM Ca2+o or bath application of A23187, a Ca2+ ionophore. In contrast, WIN55,212-2 suppressed mIPSC frequency with 2mM Ca2+o, but it became ineffective when the presynaptic Ca2+ level was lowered by perfusion with a Ca2+-free solution containing BAPTA-AM. Experiments with systematic [Ca2+]o changes revealed that mIPSC but not mEPSC regularly involved events elicited by presynaptic Ca2+ rise with 2mM Ca2+o. Importantly, Ca2+-enhancement of mEPSC and mIPSC was not attributable to activation of voltage-dependent Ca2+ channels. Activation of GABAB receptor or group III metabotropic glutamate receptor, which couple to Gi/o-protein, also preferentially suppressed Ca2+-enhanced miniature events in PCs. These results suggest that the occurrence of Ca2+-enhanced miniature events at normal [Ca2+]o determines the sensitivity to the presynaptic depression mediated by cannabinoid receptors and other Gi/o-coupled receptors in PCs. [ABSTRACT FROM AUTHOR]

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid- mediated suppression of excitatory/inhibitory synaptic transmission. Activation of Gq/11-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC)β4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCβ4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCβ4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCβ4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs. [ABSTRACT FROM AUTHOR]

Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca2+ channel α1A, a major Ca2+ channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the α1A knock-out mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers consisting of a strong climbing fiber and one or a few weaker climbing fibers, persisted in the majority of Purkinje cells and were cowired to the same somata, proximal dendrites, or both. Therefore, the lack of α1A results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca2+ channel α1A fuels heterosynaptic competition between climbing fibers and parallel fibers and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along the dendritic tree of the Purkinje cell and also establishes climbing fiber monoinnervation of individual Purkinje cells. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) stress response contributes to neuronal survival in ischemia and neurodegenerative processes. ORP 150 (oxygen-regulated protein 150)/HSP12A (heat shock protein 12A), a novel stress protein located in the ER, was markedly induced in Purkinje cells maximally at 4 - 8 d after birth, a developmental period corresponding to their vulnerability to cell death. Both terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling analysis and immunostaining using anti-activated caspase-3 antibody revealed that transgenic mice with targeted neuronal overexpression of ORP 150 (Tg ORP 150) displayed diminished cell death in the Purkinje cell layer and increased numbers of Purkinje cells up to 40 d after birth (p < 0.01), compared with those observed in heterozygous ORP 150/HSP 12A-deficient (ORP 150+/-) mice and wild-type littermates (ORP 150+/+). Cultured Purkinj e cells from Tg ORP 150 mice displayed resistance to both hypoxia- and AMPA-induced stress. Behavioral analysis, using rotor rod tasks, indicated impairment of cerebellar function in Tg OR P150 animals, consistent with the concept that enhanced survival of Purkinje cells results in dysfunction. These data suggest that ER chaperones have a pivotal role in Purkinje cell survival and death and thus may highlight the importance of ER stress in neuronal development. [ABSTRACT FROM AUTHOR]