Search

Functional neural circuits in the mature animals are shaped during postnatal development by elimination of unnecessary synapses and strengthening of necessary ones among redundant synaptic connections formed transiently around birth. In the cerebellum of neonatal rodents, excitatory synapses are formed on the somata of Purkinje cells (PCs) by climbing fibers (CFs) that originate from neurons in the contralateral inferior olive. Each PC receives inputs from multiple (~ five) CFs that have about equal synaptic strengths. Subsequently, a single CF selectively becomes stronger relative to the other CFs during the first postnatal week. Then, from around postnatal day 9 (P9), only the strongest CF (“winner” CF) extends its synaptic territory along PC dendrites. In contrast, synapses of the weaker CFs (“loser” CFs) remain on the soma and the most proximal portion of the dendrite together with somatic synapses of the “winner” CF. These perisomatic CF synapses are eliminated progressively during the second and the third postnatal weeks. From P6 to P11, the elimination proceeds independently of the formation of the synapses on PC dendrites by parallel fibers (PFs). From P12 and thereafter, the elimination requires normal PF-PC synapse formation and is presumably dependent on the PF synaptic inputs. Most PCs become mono-innervated by single strong CFs on their dendrites in the third postnatal week. In this review article, we will describe how adult-type CF mono-innervation of PC is established through these multiple phases of postnatal cerebellar development and make an overview of molecular/cellular mechanisms underlying them. [ABSTRACT FROM AUTHOR]

Progress in research on endocannabinoid signaling has greatly advanced our understanding of how it controls neural circuit excitability in health and disease. In general, endocannabinoid signaling at excitatory synapses suppresses seizures by inhibiting glutamate release. In contrast, endocannabinoid signaling promotes seizures by inhibiting GABA release at inhibitory synapses. The physiological distribution of endocannabinoid signaling molecules becomes disrupted with the development of epileptic focus in patients with mesial temporal lobe epilepsy and in animal models of experimentally induced epilepsy. Augmentation of endocannabinoid signaling can promote the development of epileptic focus at initial stages. However, at later stages, increased endocannabinoid signaling delays it and suppresses spontaneous seizures. Thus, the regulation of endocannabinoid signaling at specific synapses that cause hyperexcitability during particular stages of disease development may be effective for treating epilepsy and epileptogenesis. [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]

Neurodegenerative diseases such as spinocerebellar ataxias and autoantibody-associated disorders of the central nervous system often affect the cerebellum, resulting in motor deficits. Recent studies have revealed that most of these disorders impair type 1 metabotropic glutamate receptor (mGluR1) and/or the closely associated signaling molecules in cerebellar Purkinje cell. Since the signaling pathway triggered by mGluR1 activation in Purkinje cell plays a pivotal role in coordinated movements and motor learning, pharmacological repair of aberrant mGluR1 signaling in Purkinje cell is critical for mitigation of cerebellar symptoms. Here we review recently identified pathophysiology underlying the neurodegenerative and autoimmune diseases affecting mGluR1 signaling in Purkinje cell and possible therapeutic interventions. [ABSTRACT FROM AUTHOR]

One of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), serves as a retrograde messenger at various types of synapses throughout the brain. Upon postsynaptic activation, 2-AG is released immediately after de novo synthesis, activates presynaptic CB 1 cannabinoid receptors, and transiently suppresses neurotransmitter release. When CB 1 receptor activation is combined with some other factors such as presynaptic activity, the suppression is converted to a long-lasting form. Whereas 2-AG primarily transmits a rapid, transient, point-to-point retrograde signal, the other major endocannabinoid, anandamide, may function as a relatively slow retrograde or non-retrograde signal or as an agonist of the vanilloid receptor. The endocannabinoid system can be up- or down-regulated by a variety of physiological and environmental factors including stress, which might be clinically important. [ABSTRACT FROM AUTHOR]

Neural circuits in neonatal animals contain numerous redundant synapses that are functionally immature. During the postnatal period, unnecessary synapses are eliminated while functionally important synapses become stronger and mature. The climbing fiber (CF) to the Purkinje cell (PC) synapse is a representative model for the analysis of postnatal refinement of neuronal circuits in the central nervous system. PCs are initially innervated by multiple CFs with similar strengths around postnatal day 3 (P3). Only a single CF is selectively strengthened during P3–P7 (functional differentiation), and the strengthened CF undergoes translocation from soma to dendrites of PCs from P9 on (dendritic translocation). Following the functional differentiation, supernumerary CF synapses on the soma are eliminated, which proceeds in two distinct phases: the early phase from P7 to around P11 and the late phase from around P12 to P17. Here, we review our current understanding of cellular and molecular mechanisms of CF synapse elimination in the developing cerebellum. [ABSTRACT FROM AUTHOR]

Abstract: The Purkinje cells in the cerebellum are unique neurons that generate local and global Ca signals in response to two types of excitatory inputs, parallel fiber and climbing fiber, respectively. The spatiotemporal distribution and interaction of these synaptic inputs produce complex patterns of Ca dynamics in the Purkinje cell dendrites. The Ca signals originate from Ca influx through voltage-gated Ca channels and Ca release from intracellular stores that are mediated by the metabotropic glutamate receptor signaling pathway. These Ca signals are essential for the induction of various forms of synaptic plasticity and for controlling the input–output relationship of Purkinje cells. In this article we review Ca signaling in Purkinje cell dendrites. [Copyright &y& Elsevier]

Functional neural circuit formation during postnatal development involves massive elimination of early-formed redundant synapses and strengthening of necessary synaptic connections. In the cerebellum, one-to-one connection from a climbing fibre (CF) to a Purkinje cell (PC) is established through four distinct phases: (1) strengthening of a single CF among multiple CFs in each PC at postnatal age P3-P7 days, (2) translocation of a single strengthened CF to PC dendrites from around P9, (3) early-phase (P7 to around P11) and (4) late-phase (around P12-P17) elimination of weak CF synapses from PC somata. Mice with PC-selective deletion of the P/Q-type voltage-dependent Ca2+ channel (VDCC) exhibit severe defects in strengthening of single CFs, dendritic translocation of single CFs and CF elimination from P7. In contrast, mice with a mutation of a single allele for the GABA synthesizing enzyme GAD67 show selective impairment of CF elimination from P10. Electrophysiological and Ca2+ imaging data suggest that GABAA receptor-mediated inhibition onto PC somata from putative basket cells influences CF-induced Ca2+ transients and regulates elimination of redundant CF synapses from PC somata at P10-P16. Thus, regulation of Ca2+ influx to PCs through VDCCs is crucial for the four phases of CF synapse elimination during postnatal development. [ABSTRACT FROM AUTHOR]

Innervation of Purkinje cells (PCs) by multiple climbing fibers (CFs) is refined into mono-innervation during the first three postnatal weeks of rodents' lives. In this review article, we will integrate the current knowledge on developmental process and mechanisms of CF synapse elimination. In the 'creeper' stage of CF innervation (postnatal day 0 (P0)∼), CFs creep among PC somata to form transient synapses on immature dendrites. In the 'pericellular nest' stage (P5∼), CFs densely surround and innervate PC somata. CF innervation is then displaced to the apical portion of PC somata in the 'capuchon' stage (P9∼), and translocate to dendrites in the 'dendritic' (P12∼) stage. Along with the developmental changes in CF wiring, functional and morphological distinctions become larger among CF inputs. PCs are initially innervated by more than five CFs with similar strengths (∼P3). During P3-7 only a single CF is selectively strengthened (functional differentiation), and it undergoes dendritic translocation from P9 on (dendritic translocation). Following the functional differentiation, perisomatic CF synapses are eliminated nonselectively; this proceeds in two distinct phases. The early phase (P7-11) is conducted independently of parallel fiber (PF)-PC synapse formation, while the late phase (P12-17) critically depends on it. The P/Q-type voltage-dependent Ca2+ channel in PCs triggers selective strengthening of single CF inputs, promotes dendritic translocation of the strengthened CFs, and drives the early phase of CF synapse elimination. In contrast, the late phase is mediated by the mGluR1-Gαq-PLCβ4-PKCγ signaling cascade in PCs driven at PF-PC synapses, whose structural connectivity is stabilized and maintained by the GluRδ2-Cbln1-neurexin system. [ABSTRACT FROM AUTHOR]

Functional neural circuit formation during postnatal development involves the elimination of early-formed redundant synapses. In the neonatal mouse cerebellum, each Purkinje cell is innervated by multiple climbing fibers (CFs). Their synaptic strengths are initially uniform but one CF strengthens relative to other CFs during the first postnatal week. Then the weaker CFs are eliminated during the second postnatal week. Projections from the retina to the lateral geniculate nucleus (LGN) undergo similar developmental changes, that is the elimination of the majority of inputs and strengthening of a few afferents that remain. Recent studies have clarified that in both cerebellum and LGN, synapse refinement consists of multiple phases and that distinct types of neural activity drive synaptic remodeling in each developmental phase. [Copyright &y& Elsevier]

The article focuses on the integration of the understanding of functions of the endocannabinoid system, especially on the control of synaptic transmission in the brain. It highlights electrophysiological studies carried out on synapses of various brain regions. In addition, it discusses the regulation of synaptic transmission by endocannabinoid signaling and the arrangement of signaling molecules around synapses.

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]

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]

We studied how physiological properties of cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses change during developmental transition from multiple to mono CF innervation onto each PC. From P3 to P6, differences in the strengths of multiple CFs became larger. Around P10, each PC was either monoinnervated by one strong CF (CF-mono) or multiply innervated by one strong CF (CF-multi-S) plus a few weaker CFs (CF-multi-W). We show that simultaneous release of multiple vesicles per site occurs normally from CF-multi-S, CF-mono, and mature CFs, but less frequently from CF-multi-W and neonatal CFs. We also present evidence suggesting that weaker CFs with lower probability of multivesicular release would be withdrawn preferentially. The results suggest that differentiation into strong and weak CFs with high and low probabilities of multivesicular release precedes developmental CF synapse elimination. [Copyright &y& Elsevier]

Discusses a study which introduced the metabotropic glutamate receptor-subtype 1 (mGluR1) transgene into mGluR1-null mutant mice with a Purkinje cell (PC)-specific promoter. Determination of climbing fibers (CF) synapse could be restored by mGluR1 transgene; Analysis of the temporal relation between the footfall of one limb and that of the other limb during treadmill locomotion; Motor coordination of both limbs.

Provides information on a study on the role of phospholipase C...4 in the developing cerebellum. Materials; Methodology; Results and discussion.

Studies impaired synapse elimination during cerebellar development in protein kinase C gamma (PKCgamma) mutant mice. Morphology of PKCgamma mutant cerebellum; CF inervation during early postnatal days; Electron microscopy and morphometry; Histology; Electrophysiology.

Demonstrates that impaired motor coordination correlates with persistent multiple climbing fiber innervation in protein kinase Cgamma (PKCgamma) mutant mice. Facilitation of eyeblink conditioning; Production of PKCgamma mutant mice; Tests for motor coordination; Electrophysiology.

Examines the cerebellar characteristics of mGluR1 mutant mice. Exhibition of cerebellar symptoms; Deficient long term depression (LTD); Impairment of eyeblink response.

Shows that the inhibitors of the calcium (Ca2+)/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. CaM-KII activation occurs shortly after conditioning calcium transients; Effects of CaM-KII inhibitors of spontaneous inhibitory postsynaptic currents.

Describes the properties and possible mechanisms of rebound potentiation (RP) of GABA-receptor mediated inhibitory postsynaptic currents in cerebellar Purkinje cells. Cellular basis of motor learning in the cerebellum; Requirements needed for the induction of RP; Involvement of protein kinases in RP of inhibitory synapses.

Presents an approach to bridge the gap between cerebellar long-term depression (LTD) studied in vitro and cerebellar functions such as motor learning. Study of synaptice plasticity and learning; Three mutants in which cerebellar LTD area deficient in vitro; Cerebellar LTD as a cellular basis for discrete motor learning.

Cerebellar climbing fibers convey sensorimotor information and their errors, which are used for motor control and learning. Furthermore, they represent reward-related information. Despite such functional diversity of climbing fiber signals, it is still unclear whether each climbing fiber conveys the information of single or multiple modalities and how the climbing fibers conveying different information are distributed over the cerebellar cortex. Here we perform two-photon calcium imaging from cerebellar Purkinje cells in mice engaged in a voluntary forelimb lever-pull task and demonstrate that climbing fiber responses in 68% of Purkinje cells can be explained by the combination of multiple behavioral variables such as lever movement, licking, and reward delivery. Neighboring Purkinje cells exhibit similar climbing fiber response properties, form functional clusters, and share noise fluctuations of responses. Taken together, individual climbing fibers convey behavioral information on multiplex variables and are spatially organized into the functional modules of the cerebellar cortex. Complex spike responses of a single mouse cerebellar Purkinje cell are modulated by multiple behavioral parameters in a self-initiated lever pull task, and there may be spatial modules of these cells to mediate functions of the cerebellar cortex. [ABSTRACT FROM AUTHOR]

Masao Ito - A Visionary Neuroscientist with a Passion for the Cerebellum. [Extracted from the article]

Cerebellar Purkinje cells (PCs) of newborn rodents are innervated by multiple climbing fibers (CFs). During the first postnatal week, single CFs are strengthened relative to other CFs on the somata of individual PCs. Then, the strengthened CFs undergo translocation to PC dendrites after P9. Elimination of the weaker CFs occurs in two distinct steps, namely the early phase from P7 to around P12 and the late phase from about P12 to around P17. Our previous study demonstrates that CF synapse elimination is severely impaired in null mutant mice lacking Ca2.1, a pore-forming component of P/Q-type voltage-dependent Ca channel (VDCC). To examine the contribution of postsynaptic P/Q-type VDCC to postnatal rearrangement of CFs, we generated mice with PC-selective deletion of Ca2.1 (PC-Ca2.1 KO). We made whole-cell recordings from PCs in cerebellar slices and examined CF-mediated excitatory postsynaptic currents. We found that PC-Ca2.1 KO PCs had severe defects in selective strengthening of single CFs during the first postnatal week and subsequent CF synapse elimination from P7. Moreover, our morphological analysis revealed that multiple CFs abnormally underwent translocation to PC dendrites in PC-Ca2.1 KO mice. These results indicate that Ca influx through P/Q-type VDCC into PCs is crucial for selective strengthening of single CFs, early phase elimination and selective translocation of single strengthened CFs to PC dendrites. [ABSTRACT FROM AUTHOR]

[Display omitted] • Grin1 deletion in prospective parvalbumin interneurons reduces unitary GABA release. • Grin1 deletion in parvalbumin neurons impairs membrane properties and excitability. • No effect of Cav2.1 channel agonist on GABA release from Grin1 -deleted neurons. • Cav2.1-haploinsufficient parvalbumin neurons produce a similar GABA release deficit. • Grin1 deletion using parvalbumin promoter causes generally normal GABA release. N -methyl-D-aspartate receptor (NMDAR) hypofunction during brain development is likely to contribute to the manifestation of schizophrenia (SCZ) in young adulthood. The cellular targets of NMDAR hypofunction appear to be at least in part corticolimbic fast-spiking (FS) interneurons. However, functional alterations in parvalbumin (PV)-positive FS interneurons following NMDAR hypofunction are poorly understood. Paired patch-clamp recordings from murine cortical PV interneurons and pyramidal neurons revealed that genetic deletion of NMDAR subunit Grin1 in prospective PV interneurons before the second postnatal week impaired evoked- and synchronized-GABA release. Whereas intrinsic excitability and spiking characteristics were also disturbed by Grin1 deletion, neither restoring their excitability by K+ channel blockade nor increasing extracellular Ca2+ rescued the GABA release. GABA release was also insensitive to the Cav2.1 channel antagonist ω-agatoxin IVA. Heterozygous deletion of Cacna1a gene (encoding Cav2.1) in PV interneurons produced a similar GABA release phenotype as the Grin1 mutants. Treatment with the Cav2.1/2.2 channel agonist GV-58 augmented somatic Ca2+ currents and GABA release in Cacna1a -haploinsufficient PV interneurons, but failed to enhance GABA release in the Grin1 -deleted PV interneurons. Taken together, our results suggest that Grin1 deletion in prospective PV interneurons impairs proper maturation of membrane excitability and Cav2.1-recruited evoked GABA release. This may increase synaptic excitatory/inhibitory ratio in principal neurons, contributing to the emergence of SCZ-like phenotypes. [ABSTRACT FROM AUTHOR]

Although ghrelin and its cognate receptor growth hormone secretagogue receptor (GHSR1a) are highly localized in the hypothalamic nuclei for the regulation of metabolic states and feeding, GHSR1a is also highly localized in the hippocampus, suggesting its involvement in extra-hypothalamic functions. Indeed, exogenous application of ghrelin has been reported to improve hippocampal learning and memory. However, the underlying mechanism of ghrelin regulation of hippocampal functions is poorly understood. Here, we report ghrelin-promoted phosphorylation of GluN1 and amplified N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents in the CA1 pyramidal cells of the hippocampus in slice preparations. The ghrelin-induced responses were sensitive to a GHSR1a antagonist and inverse agonist, and were absent in GHSR1a homozygous knock-out mice. These results indicated that activation of GHSR1a was critical in the ghrelin-induced enhancement of the NMDAR function. Interestingly, heterozygous mouse hippocampi were also insensitive to ghrelin treatment, suggesting that a slight reduction in the availability of GHSR1a may be sufficient to negate the effect of ghrelin on GluN1 phosphorylation and NMDAR channel activities. In addition, NMDAR-mediated spike currents, which are of dendritic origin, were blocked by the GHSR1a antagonist, suggesting the presence of GHSR1a on the pyramidal cell dendrites in physical proximity to NMDAR. Together with our findings on the localization of GHSR1a in the CA1 region of the hippocampus, which was shown by fluorescent ghrelin binding, immunoreactivity, and enhanced green fluorescent protein reporter gene expression, we conclude that the activation of GHSR1a favours rapid modulation of the NMDAR-mediated glutamatergic synaptic transmission by phosphorylating GluN1 in the hippocampus. [ABSTRACT FROM AUTHOR]

Opinion. Presents several reports regarding the cellular mechanisms of cerebellar long-term depression (LTD). Involvement of mGluR1 in the induction of LTD; Necessity for the glutamate receptor gamma2 subunit for LTD induction; Involvement of calcium release from intracellular stores in LTD.

Group I metabotropic glutamate receptors (mGluRs) include mGluR1 and mGluR5, which are coupled to the Gq family of heterotrimeric G-proteins and readily activated by their selective agonist 3,5-dihydroxyphenilglycine (DHPG). mGluR1 and mGluR5 exhibit nearly complementary distributions spatially or temporally in the central nervous system (CNS). In adult cerebellar Purkinje cells (PCs), mGluR1 is a dominant group I mGluR and mGluR5 is undetectable. mGluR1 expression increases substantially during the first three weeks of postnatal development and remains high throughout adulthood. On the other hand, mGluR5 expression is observed during the first two postnatal weeks and then decreases. However, functional differences between mGluR1 and mGluR5 in the CNS remains to be elucidated. To address this issue, we generated "mGluR5-rescue" mice in which mGluR5 is specifically expressed in PCs in global mGluR1-knockout (KO) mice. mGluR5-rescue mice exhibited apparently normal motor coordination, developmental elimination of redundant climbing fiber (CF)-PC synapses, and delay eyeblink conditioning, which were severely impaired in mGluR1-KO mice. We concluded that mGluR5 is functionally comparable with mGluR1 in cerebellar PCs. [ABSTRACT FROM AUTHOR]

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies. [ABSTRACT FROM AUTHOR]

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies. [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]

Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. [ABSTRACT FROM AUTHOR]

Endogenous cannabinoids (endocannabinoids) serve as retrograde messengers at synapses in various regions of the brain. They are released from postsynaptic neurons and cause transient and long-lasting reduction of neurotransmitter release through activation of presynaptic cannabinoid receptors. Endocannabinoid release is induced either by increased postsynaptic Ca2+ levels or by activation of Gq/11-coupled receptors. When these two stimuli coincide, endocannabinoid release is markedly enhanced, which is attributed to the Ca2+ dependency of phospholipase Cβ (PLCβ). This Ca2+-assisted receptor-driven endocannabinoid release is suggested to participate in various forms of synaptic plasticity, including short-term associative plasticity in the cerebellum and spike-timing-dependent long-term depression in the somatosensory cortex. In these forms of plasticity, PLCβ seems to function as a coincident detector of presynaptic and postsynaptic activities. [Copyright &y& Elsevier]

Marijuana affects neural functions through the binding of its active component Δ9-THC) to cannabinoid receptors in the CNS. Recent studies have elucidated that endogenous ligands for cannabinoid receptors, endocannabinoids, serve as retrograde messengers at central synapses. Endocannabinoids are produced on demand in activity-dependent manners and released from postsynaptic neurons. The released endocannabinoids travel backward across the synapse, activate presynaptic CB1 cannabinoid receptors, and modulate presynaptic functions. Retrograde endocannabinoid signaling is crucial for certain forms of short-term and long-term synaptic plasticity at excitatory or inhibitory synapses in many brain regions, and thereby contributes to various aspects of brain function including learning and memory. Molecular identities of the CB1 receptor and enzymes involved in production and degradation of endocannabinoids have been elucidated. Anatomical studies have demonstrated unique distributions of these molecules around synapses, which provide morphological bases for the roles of endocannabinoids as retrograde messengers. CB1-knockout mice exhibit various behavioral abnormalities and multiple defects in synaptic plasticity, supporting the notion that endocannabinoid signaling is involved in various aspects of neural function. In this review article, the authors describe molecular mechanisms of the endocannabinoid-mediated synaptic modulation and its possible physiological significance. [ABSTRACT FROM AUTHOR]

Abstract: We previously demonstrated that metabotropic glutamate receptor-subtype 1 knockout [mGluR1 (−/−)] mice showed ataxic gait, deficient long-term depression and impaired synapse elimination and these phenotypes were rescued by introduction of an mGluR1 transgene with Purkinje cell-specific promoter (mGluR1-rescue mice). However, roles of mGluR1 in the adult brain remain elusive, mainly due to lack of conventional and reproducible method to block mGluR1 expression at a certain developmental stage. Here, we established a versatile mouse line, mGluR1 conditional knockout (cKO) mice using the tetracycline-controlled gene expression system to understand the roles of mGluR1 in the adult brain. The mGluR1 cKO mice express mGluR1 only in Purkinje cells and show normal motor coordination. Blockade of expression of mGluR1 in the adult mGluR1 cKO mice led to impaired motor coordination, suggesting that mGluR1 is essential for cerebellar function in mice not only during postnatal development but also in adulthood. [Copyright &y& Elsevier]

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]

Abstract: Medium spiny neurons in the dorsal striatum receive glutamatergic excitatory synaptic inputs from the cerebral cortex. These synapses undergo long-term depression that requires release of endocannabinoids from medium spiny neurons and activation of cannabinoid CB1 receptors. However, it remains unclear how cortico-striatal synapses exhibit endocannabinoid-mediated short-term suppression, which has been found in various brain regions including the hippocampus and cerebellum. Endocannabinoids are released from postsynaptic neurons by strong depolarization and resultant Ca2+ elevation or activation of postsynaptic Gq/11-coupled receptors such as group I metabotropic glutamate receptors (mGluRs) and M1/M3 muscarinic acetylcholine receptors. Moreover, endocannabioids are effectively released when weak depolarization is combined with Gq/11-coupled receptor activation. We found that muscarinic activation induced transient suppression of excitatory synaptic transmission to medium spiny neurons, which was independent of retrograde endocannabinoid signaling but was mediated directly by presynaptic muscarinic receptors. Neither postsynaptic depolarization alone nor depolarization and muscarinic activation caused suppression of cortico-striatal synapses. In contrast, activation of group I mGluRs readily suppressed cortico-striatal excitatory synaptic transmission. Furthermore, postsynaptic depolarization induced clear suppression when combined with group I mGluR activation. These results indicate that group I mGluRs but not muscarinic receptors contribute to endocannabinoid-mediated short-term suppression of cortico-striatal excitatory synaptic transmission. [Copyright &y& Elsevier]

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]

The metabotropicglutamate receptors (mGluRs) are neurotransmitter receptors important for synaptic plasticity in the brain. Here we report that native mGluR-mediated neuronal responses to glutamate are profoundly modulated by extracellular calcium (Ca2+o). In mouse cerebellar Purkinje cells (PCs), Ca2+o drastically broadened the effective dose range for glutamate analogs in which native mGluR1-mediated cation current and intracellular Ca2+ mobilization were evoked. This effect has not been observed for recombinant mGluRs expressed in the heterologous cell systems. Ca2+o also drastically augmented these native mGluR-mediated responses to the glutamate analog. These Ca2+o effects were observed in both the wild-type mice and the mutant mice expressing mGluR1 specifically in their PCs, suggesting that the native mGluR1 in the PCs but not those in other cell types are the key mediators of the effects. These findings demonstrate that Ca2+o plays an important role in regulating native mGluR-mediated neuronal responses. [Copyright &y& Elsevier]

• 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]

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for studying the local dynamics of neurotransmitters and neuromodulators. FSCV is attractive to researchers employing electrophysiological techniques because it can be performed using an electrophysiological voltage-clamp amplifier. However, the narrow test potential range of electrophysiological amplifiers (typically, ±1 V) limits testable species of analytes. Here we devised a booster that extends the test potential range. Using the booster, we could detect the oxidation current of adenosine peaking near a test potential of +1.5 V. The booster should promote combined electrophysiological and electrochemical studies of synaptic release and neural secretion. Image 1 • Fast-scan cyclic voltammetry is useful for studying neurochemical dynamics. • This measurement can be performed using an electrophysiological amplifier. • However, the amplifier's test potential range is too narrow to detect some analytes. • We devised a booster to extend the test potential range. • The booster allows electrophysiologists to analyze a variety of analytes. [ABSTRACT FROM AUTHOR]

Two-photon imaging is a major recording technique used in neuroscience. However, it suffers from several limitations, including a low sampling rate, the nonlinearity of calcium responses, the slow dynamics of calcium dyes and a low SNR, all of which severely limit the potential of two-photon imaging to elucidate neuronal dynamics with high temporal resolution. We developed a hyperacuity algorithm (HA_time) based on an approach that combines a generative model and machine learning to improve spike detection and the precision of spike time inference. Bayesian inference was performed to estimate the calcium spike model, assuming constant spike shape and size. A support vector machine using this information and a jittering method maximizing the likelihood of estimated spike times enhanced spike time estimation precision approximately fourfold (range, 2–7; mean, 3.5–4.0; 2SEM, 0.1–0.25) compared to the sampling interval. Benchmark scores of HA_time for biological data from three different brain regions were among the best of the benchmark algorithms. Simulation of broader data conditions indicated that our algorithm performed better than others with high firing rate conditions. Furthermore, HA_time exhibited comparable performance for conditions with and without ground truths. Thus HA_time is a useful tool for spike reconstruction from two-photon imaging. [ABSTRACT FROM AUTHOR]

• Motor sequence learning is impaired in mice lacking the cannabinoid CB 1 receptor. • It is also impaired in mice lacking the endocannabinoid 2-AG synthetic enzyme DGLα. • Reversal learning of sequence is impaired in both CB 1 - and DGLα-knockout mice. • 2-AG to CB 1 signaling is critically involved in reward-based motor learning. The endocannabinoid system modulates synaptic transmission, controls neuronal excitability, and is involved in various brain functions including learning and memory. 2-arachidonoylglycerol, a major endocannabinoid produced by diacylglycerol lipase-α (DGLα), is released from postsynaptic neurons, retrogradely activates presynaptic CB 1 cannabinoid receptors, and induces short-term or long-term synaptic plasticity. To examine whether and how the endocannabinoid system contributes to reward-based learning of a motor sequence, we subjected male CB 1 -knockout (KO) and DGLα-KO mice to three types of operant lever-press tasks. First, we trained mice to press one of three levers labeled A, B, and C for a food reward (one-lever task). Second, we trained mice to press the three levers in the order of A, B, and C (three-lever task). Third, the order of the levers was reversed to C, B, and A (reverse three-lever task). We found that CB 1 -KO mice and DGLα-KO mice exhibited essentially the same deficits in the operant lever-press tasks. In the one-lever task, both strains of knockout mice showed a slower rate of learning to press a lever for food. In the three-lever task, both strains of knockout mice showed a slower rate of learning of the motor sequence. In the reverse three-lever task, both strains of knockout mice needed more lever presses for reversal learning. These results suggest that the endocannabinoid system facilitates reward-based learning of a motor sequence by conferring the flexibility with which animals can switch between strategies. [ABSTRACT FROM AUTHOR]

• Monoacylglycerol lipase (MAGL) inactivation increases microglial perimeter in the cerebellum. • Cerebellar microglia activation concurs with COX-2 over-expression and motor deficits. • COX-2 inhibition prevents the cerebellar neuroinflammation caused by MAGL deletion. • MAGL depletion decreased AA and PGs levels in the cerebellum and hippocampus. • Regional differences in MAGL inhibition outcome caution endocannabinoid targeting. Monoacylglycerol lipase (MAGL) is the main enzyme implicated in the degradation of the most abundant endocannabinoid in the brain, 2-arachidonoylglycerol (2-AG), producing arachidonic acid (AA) and glycerol. MAGL pharmacological inhibition with JZL184 or genetic deletion results in an exacerbated 2-AG signaling and reduced synthesis of prostaglandins (PGs), due to the reduced AA precursor levels. We found that acute JZL184 administration, previously described to exert anti-inflammatory effects, and MAGL knockout (KO) mice display cerebellar, but not hippocampal, microglial reactivity, accompanied with increased expression of the mRNA levels of neuroinflammatory markers, such as cyclooxygenase-2 (COX-2). Notably, this neuroinflammatory phenotype correlated with relevant motor coordination impairment in the beam-walking and the footprint tests. Treatment with the COX-2 inhibitor NS398 during 5 days prevented the deficits in cerebellar function and the cerebellar microglia reactivity in MAGL KO, without affecting hippocampal reactivity. Altogether, this study reveals the brain region-specific response to MAGL inhibition, with an important role of COX-2 in the cerebellar deficits associated, which should be taken into account for the use of MAGL inhibitors as anti-inflammatory drugs. [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]