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A central hypothesis concerning brain functioning is that plasticity regulates the signal transfer function by modifying the efficacy of synaptic transmission. In the cerebellum, the granular layer has been shown to control the gain of signals transmitted through the mossy fiber pathway. Until now, the impact of plasticity on incoming activity patterns has been analyzed by combining electrophysiological recordings in acute cerebellar slices and computational modeling, unraveling a broad spectrum of different forms of synaptic plasticity in the granular layer, often accompanied by forms of intrinsic excitability changes. Here, we attempt to provide a brief overview of the most prominent forms of plasticity at the excitatory synapses formed by mossy fibers onto primary neuronal components (granule cells, Golgi cells and unipolar brush cells) in the granular layer. Specifically, we highlight the current understanding of the mechanisms and their functional implications for synaptic and intrinsic plasticity, providing valuable insights into how inputs are processed and reconfigured at the cerebellar input stage.

The neuroscientific field benefits from the conjoint evolution of experimental and computational techniques, allowing for the reconstruction and simulation of complex models of neurons and synapses. Chemical synapses are characterized by presynaptic vesicle cycling, neurotransmitter diffusion, and postsynaptic receptor activation, which eventually lead to postsynaptic currents and subsequent membrane potential changes. These mechanisms have been accurately modeled for different synapses and receptor types (AMPA, NMDA, and GABA) of the cerebellar cortical network, allowing simulation of their impact on computation. Of special relevance is short-term synaptic plasticity, which generates spatiotemporal filtering in local microcircuits and controls burst transmission and information flow through the network. Here, we present how data-driven computational models recapitulate the properties of neurotransmission at cerebellar synapses. The simulation of microcircuit models is starting to reveal how diverse synaptic mechanisms shape the spatiotemporal profiles of circuit activity and computation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Masoli, Rizza, Tognolina, Prestori and D’Angelo.)

Autism spectrum disorders (ASD) are pervasive neurodevelopmental disorders that include a variety of forms and clinical phenotypes. This heterogeneity complicates the clinical and experimental approaches to ASD etiology and pathophysiology. To date, a unifying theory of these diseases is still missing. Nevertheless, the intense work of researchers and clinicians in the last decades has identified some ASD hallmarks and the primary brain areas involved. Not surprisingly, the areas that are part of the so-called "social brain", and those strictly connected to them, were found to be crucial, such as the prefrontal cortex, amygdala, hippocampus, limbic system, and dopaminergic pathways. With the recent acknowledgment of the cerebellar contribution to cognitive functions and the social brain, its involvement in ASD has become unmistakable, though its extent is still to be elucidated. In most cases, significant advances were made possible by recent technological developments in structural/functional assessment of the human brain and by using mouse models of ASD. Mouse models are an invaluable tool to get insights into the molecular and cellular counterparts of the disease, acting on the specific genetic background generating ASD-like phenotype. Given the multifaceted nature of ASD and related studies, it is often difficult to navigate the literature and limit the huge content to specific questions. This review fulfills the need for an organized, clear, and state-of-the-art perspective on cerebellar involvement in ASD, from its connections to the social brain areas (which are the primary sites of ASD impairments) to the use of monogenic mouse models.

The functional properties of cerebellar stellate cells and the way they regulate molecular layer activity are still unclear. We have measured stellate cells electroresponsiveness and their activation by parallel fiber bursts. Stellate cells showed intrinsic pacemaking, along with characteristic responses to depolarization and hyperpolarization, and showed a marked short-term facilitation during repetitive parallel fiber transmission. Spikes were emitted after a lag and only at high frequency, making stellate cells to operate as delay-high-pass filters. A detailed computational model summarizing these physiological properties allowed to explore different functional configurations of the parallel fiber-stellate cell-Purkinje cell circuit. Simulations showed that, following parallel fiber stimulation, Purkinje cells almost linearly increased their response with input frequency, but such an increase was inhibited by stellate cells, which leveled the Purkinje cell gain curve to its 4 Hz value. When reciprocal inhibitory connections between stellate cells were activated, the control of stellate cells over Purkinje cell discharge was maintained only at very high frequencies. These simulations thus predict a new role for stellate cells, which could endow the molecular layer with low-pass and band-pass filtering properties regulating Purkinje cell gain and, along with this, also burst delay and the burst-pause responses pattern.

The cerebellum is most renowned for its role in sensorimotor control and coordination, but a growing number of anatomical and physiological studies are demonstrating its deep involvement in cognitive and emotional functions. Recently, the development and refinement of optogenetic techniques boosted research in the cerebellar field and, impressively, revolutionized the methodological approach and endowed the investigations with entirely new capabilities. This translated into a significant improvement in the data acquired for sensorimotor tests, allowing one to correlate single-cell activity with motor behavior to the extent of determining the role of single neuronal types and single connection pathways in controlling precise aspects of movement kinematics. These levels of specificity in correlating neuronal activity to behavior could not be achieved in the past, when electrical and pharmacological stimulations were the only available experimental tools. The application of optogenetics to the investigation of the cerebellar role in higher-order and cognitive functions, which involves a high degree of connectivity with multiple brain areas, has been even more significant. It is possible that, in this field, optogenetics has changed the game, and the number of investigations using optogenetics to study the cerebellar role in non-sensorimotor functions in awake animals is growing. The main issues addressed by these studies are the cerebellar role in epilepsy (through connections to the hippocampus and the temporal lobe), schizophrenia and cognition, working memory for decision making, and social behavior. It is also worth noting that optogenetics opened a new perspective for cerebellar neurostimulation in patients (e.g., for epilepsy treatment and stroke rehabilitation), promising unprecedented specificity in the targeted pathways that could be either activated or inhibited.

Spinocerebellar ataxias (SCAs) constitute a heterogeneous group of more than 40 autosomal-dominant genetic and neurodegenerative diseases characterized by loss of balance and motor coordination due to dysfunction of the cerebellum and its efferent connections. Despite a well-described clinical and pathological phenotype, the molecular and cellular events that underlie neurodegeneration are still poorly undaerstood. Emerging research suggests that mutations in SCA genes cause disruptions in multiple cellular pathways but the characteristic SCA pathogenesis does not begin until calcium signaling pathways are disrupted in cerebellar Purkinje cells. Ca 2+ signaling in Purkinje cells is important for normal cellular function as these neurons express a variety of Ca 2+ channels, Ca2+-dependent kinases and phosphatases, and Ca 2+ -binding proteins to tightly maintain Ca 2+ homeostasis and regulate physiological Ca 2+ -dependent processes. Abnormal Ca 2+ levels can activate toxic cascades leading to characteristic death of Purkinje cells, cerebellar atrophy, and ataxia that occur in many SCAs. The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells via inhibitory signals; thus, Purkinje cell dysfunction or degeneration would partially or completely impair the cerebellar output in SCAs. In the absence of the inhibitory signal emanating from Purkinje cells, DCN will become more excitable, thereby affecting the motor areas receiving DCN input and resulting in uncoordinated movements. An outstanding advantage in studying the pathogenesis of SCAs is represented by the availability of a large number of animal models which mimic the phenotype observed in humans. By mainly focusing on mouse models displaying mutations or deletions in genes which encode for Ca 2+ signaling-related proteins, in this review we will discuss the several pathogenic mechanisms related to deranged Ca 2+ homeostasis that leads to significant Purkinje cell degeneration and dysfunction.

Neuronal inhibition can be defined as a spatiotemporal restriction or suppression of local microcircuit activity. The importance of inhibition relies in its fundamental role in shaping signal processing in single neurons and neuronal circuits. In this context, the activity of inhibitory interneurons proved the key to endow networks with complex computational and dynamic properties. In the last 50 years, the prevailing view on the functional role of cerebellar cortical inhibitory circuits was that excitatory and inhibitory inputs sum spatially and temporally in order to determine the motor output through Purkinje cells (PCs). Consequently, cerebellar inhibition has traditionally been conceived in terms of restricting or blocking excitation. This assumption has been challenged, in particular in the cerebellar cortex where all neurons except granule cells (and unipolar brush cells in specific lobules) are inhibitory and fire spontaneously at high rates. Recently, a combination of electrophysiological recordings in vitro and in vivo , imaging, optogenetics and computational modeling, has revealed that inhibitory interneurons play a much more complex role in regulating cerebellar microcircuit functions: inhibition shapes neuronal response dynamics in the whole circuit and eventually regulate the PC output. This review elaborates current knowledge on cerebellar inhibitory interneurons [Golgi cells, Lugaro cells (LCs), basket cells (BCs) and stellate cells (SCs)], starting from their ontogenesis and moving up to their morphological, physiological and plastic properties, and integrates this knowledge with that on the more renown granule cells and PCs. We will focus on the circuit loops in which these interneurons are involved and on the way they generate feed-forward, feedback and lateral inhibition along with complex spatio-temporal response dynamics. In this perspective, inhibitory interneurons emerge as the real controllers of cerebellar functioning., (Copyright © 2019 Prestori, Mapelli and D’Angelo.)

Autism spectrum disorders (ASDs) are pervasive neurodevelopmental conditions that often involve mutations affecting synaptic mechanisms. Recently, the involvement of cerebellum in ASDs has been suggested, but the underlying functional alterations remained obscure. We investigated single-neuron and microcircuit properties in IB2 (Islet Brain-2) KO mice of either sex. The IB2 gene (chr22q13.3 terminal region) deletion occurs in virtually all cases of Phelan-McDermid syndrome, causing autistic symptoms and a severe delay in motor skill acquisition. IB2 KO granule cells showed a larger NMDA receptor-mediated current and enhanced intrinsic excitability, raising the excitatory/inhibitory balance. Furthermore, the spatial organization of granular layer responses to mossy fibers shifted from a "Mexican hat" to a "stovepipe hat" profile, with stronger excitation in the core and weaker inhibition in the surround. Finally, the size and extension of long-term synaptic plasticity were remarkably increased. These results show for the first time that hyperexcitability and hyperplasticity disrupt signal transfer in the granular layer of IB2 KO mice, supporting cerebellar involvement in the pathogenesis of ASD. SIGNIFICANCE STATEMENT This article shows for the first time a complex set of alterations in the cerebellum granular layer of a mouse model [IB2 (Islet Brain-2) KO] of autism spectrum disorders. The IB2 KO in mice mimics the deletion of the corresponding gene in the Phelan-McDermid syndrome in humans. The changes reported here are centered on NMDA receptor hyperactivity, hyperplasticity, and hyperexcitability. These, in turn, increase the excitatory/inhibitory balance and alter the shape of center/surround structures that emerge in the granular layer in response to mossy fiber activity. These results support recent theories suggesting the involvement of cerebellum in autism spectrum disorders., (Copyright © 2019 the authors 0270-6474/19/392383-15$15.00/0.)

Brain neurons exhibit complex electroresponsive properties - including intrinsic subthreshold oscillations and pacemaking, resonance and phase-reset - which are thought to play a critical role in controlling neural network dynamics. Although these properties emerge from detailed representations of molecular-level mechanisms in "realistic" models, they cannot usually be generated by simplified neuronal models (although these may show spike-frequency adaptation and bursting). We report here that this whole set of properties can be generated by the extended generalized leaky integrate-and-fire (E-GLIF) neuron model. E-GLIF derives from the GLIF model family and is therefore mono-compartmental, keeps the limited computational load typical of a linear low-dimensional system, admits analytical solutions and can be tuned through gradient-descent algorithms. Importantly, E-GLIF is designed to maintain a correspondence between model parameters and neuronal membrane mechanisms through a minimum set of equations. In order to test its potential, E-GLIF was used to model a specific neuron showing rich and complex electroresponsiveness, the cerebellar Golgi cell, and was validated against experimental electrophysiological data recorded from Golgi cells in acute cerebellar slices. During simulations, E-GLIF was activated by stimulus patterns, including current steps and synaptic inputs, identical to those used for the experiments. The results demonstrate that E-GLIF can reproduce the whole set of complex neuronal dynamics typical of these neurons - including intensity-frequency curves, spike-frequency adaptation, post-inhibitory rebound bursting, spontaneous subthreshold oscillations, resonance, and phase-reset - providing a new effective tool to investigate brain dynamics in large-scale simulations.

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR 1 ) play an important role in the responses to stressful challenges. Despite the well established expression of CRFR 1 in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR 1 expression in cerebellar GrCs, we used a mouse model depleted of CRFR 1 in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR 1 in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Analysis of cerebella transcriptome obtained from KO and control mice detected prominent alterations in the expression of calcium signaling pathways components. Moreover, male mice depleted of CRFR 1 specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion, or fear and anxiety-related behaviors. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning. SIGNIFICANCE STATEMENT Although it is known that the corticotropin-releasing factor type 1 receptor (CRFR 1 ) is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed at the effect of CRF on Purkinje cells at the cellular level and, to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of granular cell-specific ablation of CRFR 1 We found a profound effect on learning both at the cellular and behavioral levels without an effect on baseline motor skills., (Copyright © 2018 the authors 0270-6474/18/386751-15$15.00/0.)

Spike-timing-dependent plasticity (STDP) is a form of long-term synaptic plasticity exploiting the time relationship between postsynaptic action potentials (APs) and EPSPs. Surprisingly enough, very little was known about STDP in the cerebellum, although it is thought to play a critical role for learning appropriate timing of actions. We speculated that low-frequency oscillations observed in the granular layer may provide a reference for repetitive EPSP/AP phase coupling. Here we show that EPSP-spike pairing at 6 Hz can optimally induce STDP at the mossy fiber-granule cell synapse in rats. Spike timing-dependent long-term potentiation and depression (st-LTP and st-LTD) were confined to a ±25 ms time-window. Because EPSPs led APs in st-LTP while APs led EPSPs in st-LTD, STDP was Hebbian in nature. STDP occurred at 6-10 Hz but vanished >50 Hz or <1 Hz (where only LTP or LTD occurred). STDP disappeared with randomized EPSP/AP pairing or high intracellular Ca 2+ buffering, and its sign was inverted by GABA-A receptor activation. Both st-LTP and st-LTD required NMDA receptors, but st-LTP also required reinforcing signals mediated by mGluRs and intracellular calcium stores. Importantly, st-LTP and st-LTD were significantly larger than LTP and LTD obtained by modulating the frequency and duration of mossy fiber bursts, probably because STDP expression involved postsynaptic in addition to presynaptic mechanisms. These results thus show that a Hebbian form of STDP occurs at the cerebellum input stage, providing the substrate for phase-dependent binding of mossy fiber spikes to repetitive theta-frequency cycles of granule cell activity. SIGNIFICANCE STATEMENT Long-term synaptic plasticity is a fundamental property of the brain, causing persistent modifications of neuronal communication thought to provide the cellular basis of learning and memory. The cerebellum is critical for learning the appropriate timing of sensorimotor behaviors, but whether and how appropriate spike patterns could drive long-term synaptic plasticity remained unknown. Here, we show that this can actually occur through a form of spike-timing-dependent plasticity (STDP) at the cerebellar inputs stage. Pairing presynaptic and postsynaptic spikes at 6-10 Hz reliably induced STDP at the mossy fiber-granule cell synapse, with potentiation and depression symmetrically distributed within a ±25 ms time window. Thus, STDP can bind plasticity to the mossy fiber burst phase with high temporal precision., (Copyright © 2017 the authors 0270-6474/17/372809-15$15.00/0.)

The cerebellar microcircuit has been the work bench for theoretical and computational modeling since the beginning of neuroscientific research. The regular neural architecture of the cerebellum inspired different solutions to the long-standing issue of how its circuitry could control motor learning and coordination. Originally, the cerebellar network was modeled using a statistical-topological approach that was later extended by considering the geometrical organization of local microcircuits. However, with the advancement in anatomical and physiological investigations, new discoveries have revealed an unexpected richness of connections, neuronal dynamics and plasticity, calling for a change in modeling strategies, so as to include the multitude of elementary aspects of the network into an integrated and easily updatable computational framework. Recently, biophysically accurate "realistic" models using a bottom-up strategy accounted for both detailed connectivity and neuronal non-linear membrane dynamics. In this perspective review, we will consider the state of the art and discuss how these initial efforts could be further improved. Moreover, we will consider how embodied neurorobotic models including spiking cerebellar networks could help explaining the role and interplay of distributed forms of plasticity. We envisage that realistic modeling, combined with closed-loop simulations, will help to capture the essence of cerebellar computations and could eventually be applied to neurological diseases and neurorobotic control systems.

The cerebellum is involved in learning and memory of sensory motor skills. However, the way this process takes place in local microcircuits is still unclear. The initial proposal, casted into the Motor Learning Theory, suggested that learning had to occur at the parallel fiber-Purkinje cell synapse under supervision of climbing fibers. However, the uniqueness of this mechanism has been questioned, and multiple forms of long-term plasticity have been revealed at various locations in the cerebellar circuit, including synapses and neurons in the granular layer, molecular layer and deep-cerebellar nuclei. At present, more than 15 forms of plasticity have been reported. There has been a long debate on which plasticity is more relevant to specific aspects of learning, but this question turned out to be hard to answer using physiological analysis alone. Recent experiments and models making use of closed-loop robotic simulations are revealing a radically new view: one single form of plasticity is insufficient, while altogether, the different forms of plasticity can explain the multiplicity of properties characterizing cerebellar learning. These include multi-rate acquisition and extinction, reversibility, self-scalability, and generalization. Moreover, when the circuit embeds multiple forms of plasticity, it can easily cope with multiple behaviors endowing therefore the cerebellum with the properties needed to operate as an effective generalized forward controller.

Realistic modeling is a new advanced methodology for investigating brain functions. Realistic modeling is based on a detailed biophysical description of neurons and synapses, which can be integrated into microcircuits. The latter can, in turn, be further integrated to form large-scale brain networks and eventually to reconstruct complex brain systems. Here we provide a review of the realistic simulation strategy and use the cerebellar network as an example. This network has been carefully investigated at molecular and cellular level and has been the object of intense theoretical investigation. The cerebellum is thought to lie at the core of the forward controller operations of the brain and to implement timing and sensory prediction functions. The cerebellum is well described and provides a challenging field in which one of the most advanced realistic microcircuit models has been generated. We illustrate how these models can be elaborated and embedded into robotic control systems to gain insight into how the cellular properties of cerebellar neurons emerge in integrated behaviors. Realistic network modeling opens up new perspectives for the investigation of brain pathologies and for the neurorobotic field.

The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD) between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation.

The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research.

Mental processing is the product of the huge number of synaptic interactions that occur in the brain. It is easier to understand how brain functions can deteriorate than how they might be boosted. Lying at the border between the humanities, cognitive science and neurophysiology, some mental diseases offer new angles on this problematic issue. Despite their social deficits, autistic subjects can display unexpected and extraordinary skills in numerous fields, including music, the arts, calculation and memory. The advanced skills found in a subgroup of people with autism may be explained by their special mental functioning, in particular by their weak central coherence, one of the pivotal characteristics of the disorder. As a result of the increasing interest in autistic talent, there has recently emerged a tendency to screen any eccentric artist or scientist for traits of the autistic spectrum. Following this trend, we analyze the eccentricity of the popular pianist Glenn Gould and briefly discuss the major functional hypotheses on autistic hyperfunctioning, advancing proposals for functional testing. In particular, the potential involvement of rhythm-entrained systems and cerebro-cerebellar loops opens up new perspectives for the investigation of autistic disorders and brain hyperfunctioning.

Sensory stimulation conveys spike discharges of variable frequency and duration along the mossy fibres of cerebellum raising the question of whether and how these patterns determine plastic changes at the mossy fibre-granule cell synapse. Although various combinations of high-frequency bursts and membrane depolarization can induce NMDA receptor-dependent long-term depression (LTD) and long-term potentiation (LTP), the effect of different discharge frequencies remained unknown. Here we show that low-frequency mossy fibre stimulation (100 impulses1 Hz) induces mGlu receptor-dependent LTD. For various burst frequencies, the plasticity-[Ca(2+)](i) relationship was U-shaped resembling the Bienenstok-Cooper-Munro (BCM) learning rule. Moreover, LTD expression was associated with increased paired-pulse ratio, coefficient of variation and failure rate, and with a decrease in release probability, therefore showing changes opposite to those characterizing LTP. The plasticity-[Ca(2+)](i) relationship and the changes in neurotransmitter release measured by varying induction frequencies were indistinguishable from those obtained by varying high-frequency burst duration. These results suggest that different glutamate receptors converge onto a final common mechanism translating the frequency and duration of mossy fibre discharges into a regulation of the LTP/LTD balance, which may play an important role in adapting spatio-temporal signal transformations at the cerebellar input stage.

Although the role of abnormal prion protein (PrP) conformation in generating infectious brain diseases (transmissible spongiform encephalopathy) has been recognized, the function of PrP in the normal brain remains mostly unknown. In this investigation, we considered the effect of PrP gene knock-out (PrP(0/0)) on cerebellar neural circuits and in particular on granule cells, which show intense PrP expression during development and selective affinity for PrP. At the third postnatal week, when PrP expression would normally attain mature levels, PrP(0/0) mice showed low performance in the accelerating rotarod and runway tests and the functioning of 40% of granule cells was abnormal. Spikes were slow, nonovershooting, and nonrepetitive in relation with a reduction in transient inward and outward membrane currents, and also the EPSPs and EPSCs had slow kinetics. Overall, these alterations closely resembled an immature phenotype. Moreover, in slow-spiking PrP(0/0) granule cells, theta-burst stimulation was unable to induce any long-term potentiation. This profound impairment in synaptic excitation and plasticity was associated with a protracted proliferation of granule cells and disappeared at P40-P50 along with the recovery of normal motor behavior (Büeler et al., 1992). These results suggest that PrP plays an important role in granule cell development eventually regulating cerebellar network formation and motor control.

Variations in intracellular calcium concentration ([Ca2+]i) provide a critical signal for synaptic plasticity. In accordance with Hebb's postulate (Hebb, 1949), an increase in postsynaptic [Ca2+]i can induce bidirectional changes in synaptic strength depending on activation of specific biochemical pathways (Bienenstock et al., 1982; Lisman, 1989; Stanton and Sejnowski, 1989). Despite its strategic location for signal processing, spatiotemporal dynamics of [Ca2+]i changes and their relationship with synaptic plasticity at the cerebellar mossy fiber (mf)-granule cell (GrC) relay were unknown. In this paper, we report the plasticity/[Ca2+]i relationship for GrCs, which are typically activated by mf bursts (Chadderton et al., 2004). Mf bursts caused a remarkable [Ca2+]i increase in GrC dendritic terminals through the activation of NMDA receptors, metabotropic glutamate receptors (probably acting through IP3-sensitive stores), voltage-dependent calcium channels, and Ca2+-induced Ca2+ release. Although [Ca2+]i increased with the duration of mf bursts, long-term depression was found with a small [Ca2+]i increase (bursts <250 ms), and long-term potentiation (LTP) was found with a large [Ca2+]i increase (bursts >250 ms). LTP and [Ca2+]i saturated for bursts >500 ms and with theta-burst stimulation. Thus, bursting enabled a Ca2+-dependent bidirectional Bienenstock-Cooper-Munro-like learning mechanism providing the cellular basis for effective learning of burst patterns at the input stage of the cerebellum.

In the last decade, the physiology of cerebellar neurons and synapses has been extended to a considerable extent. We have found that the mossy fiber-granule cell relay can generate a complex form of long-term potentiation (mf-GrC LTP) following high-frequency mf discharge. Induction. Mf-GrC LTP depends on NMDA and mGlu receptor activation, intracellular Ca(2+) increase, PKC activation, and NO production. The preventative action of intracellular agents (BAPTA, PKC-inhibitors) and of membrane hyperpolarization, and the correlated increase in intracellular Ca(2+) observed using fluorescent dyes, indicate that induction occurs postsynaptically. Expression. Expression includes three components: (a) an increase of synaptic currents, (b) an increase of intrinsic excitability in GrC, and (c) an increase of intrinsic excitability in mf terminals. Based on quantal analysis, the EPSC increase is mostly explained by enhanced neurotransmitter release. NO is a candidate retrograde neurotransmitter which could determine both presynaptic current changes and LTP. NO cascade blockers inhibit both presynaptic current changes and LTP. The increase in intrinsic excitability involves a raise in apparent input resistance in the subthreshold region and a spike threshold reduction. Together with other forms of cerebellar plasticity, mf-GrC LTP opens new hypothesis on how the cerebellum processes incoming information.

During long-term potentiation (LTP) at mossy fibre-granule cell synapses in rat cerebellum synaptic transmission and granule cell intrinsic excitability are enhanced. Although it is clear that changes in granule cell excitability are mediated postsynaptically, there is as yet no direct evidence for the site and mechanism of changes in transmission. To approach this problem, evoked postsynaptic currents (EPSCs) and miniature synaptic currents (mEPSCs) were recorded by patch-clamp in cerebellar slices obtained from P17-P23 rats. LTP was induced by theta-burst stimulation paired with depolarization. During LTP, the EPSCs showed a significant decrease in the coefficient of variation (CV; 28.9 +/- 5.2%, n= 8; P < 0.002), the number of failures (87.1 +/- 41.9%, n= 8; P < 0.04), and the paired-pulse ratio (PPR; 25.5 +/- 4.1% n= 5; P < 0.02). Similar changes were observed by increasing neurotransmitter release (extracellular solutions with high Ca(2+)/Mg(2+) ratio), whereas increases in CV, numbers of failures and PPR occurred when release was decreased (extracellular solutions with low Ca(2+)/Mg(2+) ratio; 10 microm Cl-adenosine). No changes followed modifications of postsynaptic holding potentials, while CV and failures were reduced when the number of active synapses was increased. LTP was prevented by use of solutions with high Ca(2+)/Mg(2+) ratio. Moreover, LTP and the associated CV decrease were observed in the spillover-mediated component of AMPA EPSCs and in NMDA EPSCs. During LTP, mEPSCs did not change in amplitude or variability but significantly increased in frequency (47.6 +/- 16%, n= 4; P < 0.03). By binomial analysis changes in EPSCs were shown to be due to increased release probability (from 0.6 +/- 0.08 to 0.73 +/- 0.06, n= 7; P < 0.02) with a constant number of three to four releasing sites. These observations provide evidence for increased neurotransmitter release during LTP at mossy fibre-granule cell synapses.

Nitric oxide (NO) is a candidate retrograde messenger in long-term potentiation (LTP). The NO metabolic pathway is expressed in the cerebellar granule cell layer but its physiological role remained unknown. In this paper we have investigated the role of NO in cerebellar mossy fiber-granule cell LTP, which has postsynaptic N-methyl-d-aspartate (NMDA) receptor-dependent induction. Pre- and postsynaptic current changes were simultaneously measured by using extracellular focal recordings, and NO release was monitored with an electrochemical probe in P21 rat cerebellar slices. High-frequency mossy fiber stimulation induced LTP and caused a significant NO release (6.2 +/- 2.8 nM; n = 5) in the granular layer that was dependent on NMDA receptor as well as on nitric oxide synthase (NOS) activation. Preventing NO production by perfusing the NOS inhibitor 100 microM NG-nitro-l-arginine (L-NNA), blocking extracellular NO diffusion by 10 microM MbO2, or inhibiting the NO target guanylyl cyclase (sGC) with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-dione (ODQ) prevented LTP. Moreover, the NO donor 10 microM 2-(N,N-diethylamino)-diazenolate-2-oxide.Na (DEA-NO) induced LTP, which was mutually occlusive with LTP generated by high-frequency stimulation, prevented by ODQ, and insensitive to NMDA channel blockade (50 microM APV + 25 microM 7-Cl-kyn) or interruption of mossy fiber stimulation. Thus NO is critical for LTP induction at the cerebellar mossy fiber-granule cell relay. Interestingly, LTP manipulations were accompanied by consensual changes in the presynaptic current, suggesting that NO acts as a retrograde signal-enhancing presynaptic terminal excitability.

The involvement of presynaptic mechanisms in the expression of long-term potentiation (LTP), an enhancement of synaptic transmission suggested to take part in learning and memory in the mammalian brain, has not been fully clarified. Although evidence for enhanced vesicle cycling has been reported, it is unknown whether presynaptic terminal excitability could change as has been observed in invertebrate synapses. To address this question, we performed extracellular focal recordings in cerebellar slices. The extracellular current consisted of a pre- (P(1)/N(1)) and postsynaptic (N(2)/SN) component. In ~50% of cases, N(1) could be subdivided into N(1a) and N(1b). Whereas N(1a) was part of the fiber volley (P(1)/N(1a)), N(1b) corresponded to a Ca(2+)-dependent component accounting for 40-50% of N(1), which could be isolated from individual mossy fiber terminals visualized with fast tetramethylindocarbocyanine perchlorate (DiI). The postsynaptic response, given its timing and sensitivity to glutamate receptor antagonists [N(2) was blocked by 10 microM [1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) and SN by 100 microM APV +50 microM 7-Cl-kyn], corresponded to granule cell excitation. N(2) and SN could be reduced by 1) Ca(2+) channel blockers, 2) decreasing the Ca(2+) to Mg(2+) ratio, 3) paired-pulse stimulation, and 4) adenosine receptor activation. However, only the first two manipulations, which modify Ca(2+) influx, were associated with N(1) (or N(1b)) reduction. LTP was induced by theta-burst mossy fiber stimulation (8 trains of 10 impulses at 100 Hz separated by 150-ms pauses). Interestingly, during LTP, both N(1) (or N(1b)) and N(2)/SN persistently increased, whereas P(1) (or P(1)/N(1a)) did not change. Average changes were N(1) = 38.1 +/- 31.9, N(2) = 49.6 +/- 48.8, and SN = 59.1 +/- 35.5%. The presynaptic changes were not observed when LTP was prevented by synaptic inhibition, by N-methyl-D-aspartate and metabotropic glutamate receptor blockage, or by protein kinase C blockage. Moreover, the presynaptic changes were sensitive to Ca(2+) channel blockers (1 mM Ni(2+) and 5 microM omega-CTx-MVIIC) and occluded by K(+) channel blockers (1 mM tetraethylammmonium). Thus regulation of presynaptic terminal excitability may take part in LTP expression at a central mammalian synapse.