Search

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD. [ABSTRACT FROM AUTHOR]

Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1‐over‐expressing mice represent an animal model of early‐onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1‐over‐expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1‐over‐expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate‐aspartate transporter. Extraordinarily slow and small climbing fiber (CF)‐mediated excitatory post‐synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20‐P28 when BG ectopia was severe, but not at P9‐P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1‐over‐expressing period successfully generated mice with and without BG ectopia, depending on the over‐expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Open Practices: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14199. Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts. In the cerebellum, Mlc1 is highly expressed in Bergmann glia (BG), which is one of the astrocyte associated with Purkinje cells (PCs). Here, we found that Mlc1 over‐expression in astrocyte resulted in the BG ectopia and insufficient association with PCs, and we revealed the critical time window inducing BG ectopia. Once BG impairments were established, synaptic wiring on PCs were affected by glutamate spillover via the insufficient BG association. Our results indicated that time course of BG cytodifferentiation is one of the crucial determinant for synaptic wiring on PCs. Cover Image for this issue: doi: 10.1111/jnc.14199. [ABSTRACT FROM AUTHOR]

Elimination of early-formed redundant synapses during postnatal development is essential for functional neural circuit formation. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs). During postnatal development, a single CF is selectively strengthened in each PC and becomes a “winner” CF that is presumed to remain into adulthood, whereas the other “loser” CFs are eliminated. These developmental changes are dependent on neural activity and signal cascades in postsynaptic PCs. Several molecules essential for CF synapse elimination have been identified in postsynaptic PCs. Importantly, we have recently uncovered that Semaphorin3A (Sema3A) and Semaphorin7A (Sema7A) derived from postsynaptic PCs act retrogradely onto presynaptic CFs and regulate CF synapse elimination. We demonstrate that Sema3A strengthens and maintains CF synapses from postnatal day 8 (P8) to P18 and opposes the force of CF elimination. In contrast, Sema7A facilitates elimination of weaker CFs from PC somata after P15. In the continuing studies, we searched for molecules that mediate these retrograde semaphorin signals in presynaptic CFs. This short article describes how Sema3A strengthens and maintains, whereas Sema7A promotes elimination of CF synapses through respective receptors and downstream molecules in presynaptic CFs during postnatal cerebellar development. [ABSTRACT FROM AUTHOR]

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]

Cerebellar cortex has an elaborate rostrocaudal organization comprised of numerous microzones. Purkinje cells (PCs) in the same microzone show synchronous activity of complex spikes (CSs) evoked by excitatory inputs from climbing fibers (CFs) that arise from neurons in the inferior olive (IO). The synchronous CS activity is considered to depend on electrical coupling among IO neurons and anatomical organization of the olivo-cerebellar projection. To determine how the CF-PC wiring contributes to the formation of microzone, we examined the synchronous CS activities between neighboring PCs in the glutamate receptor d2 knockout (GluD2 KO) mouse in which exuberant surplus CFs make ectopic innervations onto distal dendrites of PCs. We performed in vivo two-photon calcium imaging for PC populations to detect CF inputs. Neighboring PCs in GluD2 KO mice showed higher synchrony of calcium transients than those in wild-type (control) mice. Moreover, the synchrony in GluD2 KO mice hardly declined with mediolateral separation between PCs up to ~200 µm, which was in marked contrast to the falloff of the synchrony in control mice. The enhanced synchrony was only partially affected by the blockade of gap junctional coupling. On the other hand, transverse CF collaterals in GluD2 KO mice extended beyond the border of microzone and formed locally clustered ectopic synapses onto dendrites of neighboring PCs. Furthermore, PCs in GluD2 KO mice exhibited clustered firing (Cf), the characteristic CF response that was not found in PCs of wild-type mice. Importantly, Cf was often associated with localized calcium transients in distal dendrites of PCs, which are likely to contribute to the enhanced synchrony of calcium signals in GluD2 KO mice. Thus, our results indicate that CF signals in GluD2 KO mice propagate across multiple microzones, and that proper formation of longitudinal olivo-cerebellar projection is essential for the spatiotemporal organization of CS activity in the cerebellum. [ABSTRACT FROM AUTHOR]

Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca2+-induced Ca2+ release and the subsequent opening of small-conductance Ca2+-activated K+ (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber–PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca2+-mediated communication between voltage-gated Ca2+ channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions. [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]

Since Purkinje cells are the sole output neurons of the cerebellar cortex, the postsynaptic integration of excitatory and inhibitory synaptic inputs in this cell type is a pivotal step for cerebellar motor information processing. In Purkinje cells, G i/o protein‐coupled B‐type γ‐aminobutyric acid receptor (GABA B R) is expressed at the annuli of the dendritic spines that are innervated by the glutamatergic terminals of parallel fibers. The subcellular localization of GABA B R suggests the possibility of postsynaptic interplay between GABA B R and glutamate signaling. It has recently been demonstrated that GABA B R indeed modulates α amino‐3‐hydroxy‐5‐methyl‐4‐isoxalone propionate‐type ionotropic glutamate receptor (AMPAR)‐mediated and type‐1 metabotropic glutamate receptor (mGluR1)‐mediated signaling. Interestingly, GABA B R exerts modulatory actions not only via the classical G i/o protein‐dependent signaling cascade but also via a G i/o protein‐independent interaction between GABA B R and mGluR1. In this review, we compare the physiological nature, underlying mechanisms, and possible functional significance of these modulatory actions of GABA B R. [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]

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]

Abstract: Cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses in rodents provides a good model to study mechanisms underlying postnatal development of synaptic functions and elimination of redundant synapses in the central nervous system. At birth, each PC is innervated by multiple CFs. Then, single CF input is selected, matured and strengthened, while surplus CFs are eliminated. By the end of the third postnatal week, most PCs become innervated by single CFs. This up-date article aims to provide an overview of recent studies on the mechanisms of this process. [Copyright &y& Elsevier]

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]

Abstract Long-term depression (LTD) at parallel fibre–Purkinje cell synapse of the cerebellum is thought to be a cellular substrate for motor learning. LTD requires activation of metabotropic glutamate receptor subtype 1 (mGluR1) and its downstream signalling pathways, which invariably involves phospholipase Cβs (PLCβs). PLCβs consist of four isoforms (PLCβ1–4) among which PLCβ4 is the major isoform in most Purkinje cells in the rostral cerebellum (lobule 1 to the rostral half of lobule 6). We studied mutant mice deficient in PLCβ4, and found that LTD was deficient in the rostral but not in the caudal cerebellum of the mutant. Basic properties of parallel fibre–Purkinje cell synapses and voltage-gated Ca2+ channel currents appeared normal. The mGluR1-mediated Ca2+ release induced by repetitive parallel fibre stimulation was absent in the rostral cerebellum of the mutant, suggesting that their LTD lesion was due to the defect in the mGluR1-mediated signalling in Purkinje cells. Importantly, the eyeblink conditioning, a simple form of discrete motor learning, was severely impaired in PLCβ4 mutant mice. Wild-type mice developed the conditioned eyeblink response, when pairs of the conditioned stimulus (tone) and the unconditioned stimulus (periorbital shock) were repeatedly applied. In contrast, PLCβ4 mutant mice could not learn the association between the conditioned and unconditioned stimuli, although their behavioural responses to the tone or to the periorbital shock appeared normal. These results strongly suggest that PLCβ4 is essential for LTD in the rostral cerebellum, which may be required for the acuisition of the conditioned eyeblink response. [ABSTRACT FROM AUTHOR]

Abstract Functional neural circuit formation includes the process by which redundant synaptic connections formed earlier during development are subsequently eliminated. We report that insulin-like growth factor I (IGF-I) is a candidate factor that influences the developmental transition from multiple to mono innervation of cerebellar Purkinje cells (PCs) by climbing fibres (CFs). Continuous local application of exogenous IGF-I to the mouse cerebellum by means of ethylene-vinyl acetate copolymer (Elvax) significantly increased the degree of multiple CF innervation, when the IGF-I containing Elvax was implanted at postnatal day 8 (P8). In contrast, the IGF-I application starting at P12 had no effect on CF innervation. Conversely, continuous local application of antisera against IGF-I and its receptor significantly decreased the degree of multiple CF innervation when the application started at P8. We found that chronic treatment of exogenous IGF-I from P8 significantly enhanced the CF-mediated excitatory postsynaptic currents (CF-EPSCs). This effect was manifest for the smaller CF-EPSCs but not for the largest CF-EPSC of the multiple-innervated PCs. Conversely, chronic application of antisera from P8 caused attenuation of the largest CF-EPSCs. Other parameters for basic synaptic functions and cerebellar morphology were largely normal after the IGF-I or antisera treatment. These results suggest that IGF-I enhances the strength of developing CF synapses and may promote their survival, whereas the shortage of IGF-I impairs the development of CF synapses and, as a result, may facilitate their elimination. Thus, IGF-I is a potentially important factor among various signalling molecules that can influence CF synapse elimination during cerebellar development. [ABSTRACT FROM AUTHOR]

The cerebellum is essential for the control, coordination, and learning of movements, and for certain aspects of cognitive function. Purkinje cells are the sole output neurons in the cerebellar cortex and therefore play crucial roles in the diverse functions of the cerebellum. The type 1 metabotropic glutamate receptor (mGluR1) is prominently enriched in Purkinje cells and triggers downstream signaling pathways that are required for functional and structural plasticity, and for synaptic responses. To understand how mGluR1 contributes to cerebellar functions, it is important to consider not only the operational properties of this receptor, but also its spatial organization and the molecular interactions that enable its proper functioning. In this review, we highlight how mGluR1 and its related signaling molecules are organized into tightly coupled microdomains to fulfill physiological functions. We also describe emerging evidence that altered mGluR1 signaling in Purkinje cells underlies cerebellar dysfunction in ataxias of human patients and mouse models. • mGluR1 signaling in Purkinje cells is crucial for various cerebellar functions. • mGluR1 triggers intracellular pathways for neural plasticity and modulation. • mGluR1 and its interacting partners are arranged into tightly coupled microdomains. • mGluR1 signaling is a promising therapeutic target for cerebellar dysfunctions. [ABSTRACT FROM AUTHOR]

• PLCβ3-knockdown in PCs impaired the late phase of CF synapse elimination in Aldoc (+) compartments of the cerebellum. • Deletion of Aldoc from PCs did not affect CF synapse elimination. • The knockdown of PLCβ3 increased somatic CF synapses and reduced CF dendritic translocation. In the cerebellum of neonatal mice, multiple climbing fibers (CFs) form excitatory synapses on each Purkinje cell (PC). Only one CF is strengthened in each PC from postnatal day 3 (P3) to P7, whereas the other weaker CFs are eliminated progressively from ∼P7 to ∼P11 (early phase of CF elimination) and from ∼P12 to ∼P17 (late phase of CF elimination). Type 1 metabotropic glutamate receptor (mGluR1) triggers a canonical pathway in PCs for the late phase of CF elimination. Among downstream signaling molecules of mGluR1, phospholipase C β3 (PLCβ3) and β4 (PLCβ4) are expressed complementarily in PCs of aldolase C (Aldoc)-positive (+) and Aldoc-negative (−) cerebellar compartments, respectively. PLCβ4 is reported to mediate the late phase of CF elimination in the anterior half of the cerebellar vermis which corresponds to the Aldoc (−) region. However, roles of PLCβ3 and Aldoc in CF synapse elimination are unknown. Here, we investigated CF innervation of PCs in Aldoc-tdTomato knock-in mice that underwent lentivirus-mediated knockdown (KD) of PLCβ3 in PCs during postnatal development. By recording CF-mediated excitatory postsynaptic currents from PCs and immunostaining CF synaptic terminals, we found that significantly higher percentage of PCs with PLCβ3-KD remained multiply innervated by CFs in Aldoc (+) compartments after P12, which was accompanied by impaired elimination of somatic CF synapses and reduced dendritic CF translocation. In contrast, deletion of Aldoc had no effect on CF synapse elimination. These results suggest that PLCβ3 is required for the late phase of CF elimination in Aldoc (+) PCs. [ABSTRACT FROM AUTHOR]

Abstract: 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 Ca2+ transients in PCs, and THL did not suppress cannabinoid sensitivity of PFs. Moreover, DSE at PF–PC synapses was absent in CB1 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. [Copyright &y& Elsevier]

Abstract: The proper regulation of microtubule (MT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a MT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs. [Copyright &y& Elsevier]

Summary Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single “winner” CFs that survive synapse elimination. [ABSTRACT FROM AUTHOR]