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GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b -/- mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b -/- PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b -/- mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b -/- mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b -/- mice. In Gprc5b -/- mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning., (Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum. SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions., (Copyright © 2018 the authors 0270-6474/18/386131-15$15.00/0.)

Adaptations of vestibulo-ocular and optokinetic response eye movements have been studied as an experimental model of cerebellum-dependent motor learning. Several previous physiological and pharmacological studies have consistently suggested that the cerebellar flocculus (FL) Purkinje cells (P-cells) and the medial vestibular nucleus (MVN) neurons targeted by FL (FL-targeted MVN neurons) may respectively maintain the memory traces of short- and long-term adaptation. To study the basic structures of the FL-MVN synapses by light microscopy (LM) and electron microscopy (EM), we injected green florescence protein (GFP)-expressing lentivirus into FL to anterogradely label the FL P-cell axons in C57BL/6J mice. The FL P-cell axonal boutons were distributed in the magnocellular MVN and in the border region of parvocellular MVN and prepositus hypoglossi (PrH). In the magnocellular MVN, the FL-P cell axons mainly terminated on somata and proximal dendrites. On the other hand, in the parvocellular MVN/PrH, the FL P-cell axonal synaptic boutons mainly terminated on the relatively small-diameter (< 1 μm) distal dendrites of MVN neurons, forming symmetrical synapses. The majority of such parvocellular MVN/PrH neurons were determined to be glutamatergic by immunocytochemistry and in-situ hybridization of GFP expressing transgenic mice. To further examine the spatial relationship between the synapses of FL P-cells and those of vestibular nerve on the neurons of the parvocellular MVN/PrH, we added injections of biotinylated dextran amine into the semicircular canal and anterogradely labeled vestibular nerve axons in some mice. The MVN dendrites receiving the FL P-cell axonal synaptic boutons often closely apposed vestibular nerve synaptic boutons in both LM and EM studies. Such a partial overlap of synaptic boutons of FL P-cell axons with those of vestibular nerve axons in the distal dendrites of MVN neurons suggests that inhibitory synapses of FL P-cells may influence the function of neighboring excitatory synapses of vestibular nerve in the parvocellular MVN/PrH neurons., Competing Interests: The authors have declared that no competing interests exist.

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)-PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1(flox/flox)) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF-PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1(flox/flox) mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF-PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.

• Studies of ocular reflex adaptations support the cerebellar learning hypothesis. • Purkinje cell synapse plasticity driven by climbing fiber inputs induces adaptations. • The memory of adaptation is initially formed in cerebellar flocculus Purkinje cells. • Repetition of adaptations consolidate the memory of adaptations in vestibular nuclei. • The hypothesis includes integrative functions operated by the cerebrocerebellar loop. Masao Ito proposed a cerebellar learning hypothesis with Marr and Albus in the early 1970s. He suggested that cerebellar flocculus (FL) Purkinje cells (PCs), which directly inhibit the vestibular nuclear neurons driving extraocular muscle motor neurons, adaptively control the horizontal vestibulo-ocular reflex (HVOR) through the modification of mossy and parallel fiber-mediated vestibular responsiveness by visual climbing fiber (CF) inputs. Later, it was suggested that the same FL PCs adaptively control the horizontal optokinetic response (HOKR) in the same manner through the modification of optokinetic responsiveness in rodents and rabbits. In 1982, Ito and his colleagues discovered the plasticity of long-term depression (LTD) at parallel fiber (PF)–PC synapses after conjunctive stimulation of mossy or parallel fibers with CFs. Long-term potentiation (LTP) at PF–PC synapses by weak PF stimulation alone was found later. Many lines of experimental evidence have supported their hypothesis using various experimental methods and materials for the past 50 years by many research groups. Although several controversial findings were presented regarding their hypothesis, the reasons underlying many of them were clarified. Today, their hypothesis is considered as a fundamental mechanism of cerebellar learning. Furthermore, it was found that the memory of adaptation is transferred from the FL to vestibular nuclei for consolidation by repetition of adaptation through the plasticity of vestibular nuclear neurons. In this article, after overviewing their cerebellar learning hypothesis, I discuss possible roles of LTD and LTP in gain-up and gain-down HVOR/HOKR adaptations and refer to the expansion of their hypothesis to cognitive functions. [ABSTRACT FROM AUTHOR]

Cerebellar Purkinje cells (PCs) project their axon collaterals to underneath of the PC layer and make GABAergic synaptic contacts with globular cells, a subgroup of Lugaro cells. GABAergic transmission derived from the PC axon collaterals is so powerful that it could inhibit globular cells and regulate their firing patterns. However, the physiological properties and implications of the GABAergic synapses on globular cells remain unknown. Using whole-cell patch-clamp recordings from globular cells in the mouse cerebellum, we examined the monoaminergic modulation of GABAergic inputs to these cells. Application of either serotonin (5-HT) or noradrenaline (NA) excited globular cells, thereby leading to their firing. The 5-HT- and NA-induced firing was temporally confined and attenuated by GABAergic transmission, although 5-HT and NA exerted an inhibitory effect on the release of GABA from presynaptic terminals of PC axon collaterals. Agonists for 5-HT 1B receptors and α 2 -adrenoceptors mimicked the 5-HT- and NA-induced suppression of GABAergic activity. Through their differential modulatory actions on the cerebellar inhibitory neural circuits, 5-HT facilitated PC firing, whereas NA suppressed it. These results indicate that 5-HT and NA regulate the membrane excitability of globular cells and PCs through their differential modulation of not only the membrane potential but also GABAergic synaptic circuits. Monoaminergic modulation of the neural connections between globular cells and PCs could play a role in cerebellar motor coordination. [ABSTRACT FROM AUTHOR]

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice-patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti-motilin receptor antibody. Bath application of motilin (0.1 μ m) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose-dependent manner ( Kd, 0.03 μ m). The motilin action on spontaneous action potentials was blocked by apamin (100 n m), a blocker of small-conductance Ca2+-activated K+ channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate-γ-S (1 m m) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild-type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ-aminobutyric acid transporter-Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small-conductance Ca2+-activated K+ channels and postsynaptic γ-aminobutyric acid receptors through heterotrimeric guanosine triphosphate-binding protein-coupled receptor in a group of glutamatergic MVNs. [ABSTRACT FROM AUTHOR]

Precise gain and timing control is the goal of cerebellar motor learning. Because the basic neural circuitry of the cerebellum is homogeneous throughout the cerebellar cortex, a single computational mechanism may be used for simultaneous gain and timing control. Although many computational models of the cerebellum have been proposed for either gain or timing control, few models have aimed to unify them. In this paper, we hypothesize that gain and timing control can be unified by learning of the complete waveform of the desired movement profile instructed by climbing fiber signals. To justify our hypothesis, we adopted a large-scale spiking network model of the cerebellum, which was originally developed for cerebellar timing mechanisms to explain the experimental data of Pavlovian delay eyeblink conditioning, to the gain adaptation of optokinetic response (OKR) eye movements. By conducting large-scale computer simulations, we could reproduce some features of OKR adaptation, such as the learning-related change of simple spike firing of model Purkinje cells and vestibular nuclear neurons, simulated gain increase, and frequency-dependent gain increase. These results suggest that the cerebellum may use a single computational mechanism to control gain and timing simultaneously. [ABSTRACT FROM AUTHOR]

In this study, we show the crucial roles of lipid signaling in longterm depression (LTD), that is, synaptic plasticity prevailing in cerebellar Purkinje cells. In mouse brain slices,, we found that cPLA2a knockout blocked LTD induction, which was rescued by replenishing arachidonic acid (AA) or prostaglandin (PG) D2 or E2. Moreover, cyclooxygenase (COX)-2 inhibitors block LTD, which is rescued by supplementing PGD2/E2. The blockade or rescue occurs when these reagents are applied within a time window of 5-15 mm following the onset of LTD-inducing stimulation. Furthermore, PGD2/E2 facilitates the chemical induction of LTD by a PKC activator but is unable to rescue the LTD blocked by a PKC inhibitor. We conclude that PGD2/E2 mediates LTD jointly with PKC, and suggest possible pathways for their interaction. Finally, we demonstrate in awake mice that cPLA2α deficiency or COX-2 inhibition attenuates short-term adaptation of optokinetic eye movements, supporting the view that LTD underlies motor learning. [ABSTRACT FROM AUTHOR]

The pontine nuclei relay information derived from the cerebral cortex to the cerebellum. In addition to the motor command signals generated in the motor cortex, the cerebellum may generate motor command signals independent of the cerebral cortex using pontine nuclei-mediated signals. The cerebellar motor command signals generated in the vermis-medial cerebellar nuclear system may directly drive peripheral motoneurons in simple and autonomic movements. Those generated in the hemisphere-lateral cerebellar nuclear system, which are used in complicated movements, may not only drive the premotor or motor nuclei but may also be fed back to the cerebellum through the parvocellular red nucleus-inferior olive pathway, and may be compared with the motor command signals generated in the cerebral cortex. The long-term depression of parallel fiber-Purkinje cell synapses may be utilized in optimizing these cerebellar motor command signals. Voluntary movements may be executed through cooperation of the cerebellum- and cerebrum-generated motor command signals. [ABSTRACT FROM AUTHOR]

Abstract: Adaptation of mouse horizontal optokinetic response (HOKR) eye movement provides an experimental model for cerebellum-dependent motor learning. Our previous study revealed that the memory trace of HOKR adaptation is initially encoded in the cerebellar flocculus after hours of optokinetic training, and transferred to the vestibular nuclei to be consolidated to long-term motor memory after days of training . To reveal how the cerebellar cortex operates in the transfer of the memory trace of adaptation, we examined the effects of shutdown of the cerebellar cortex after daily training. Three groups of mice received 1h of optokinetic training daily for 4 days, and showed similar amounts of adaptation after the end of 1h of training throughout 4 days. However, in the mice which daily received bilateral floccular muscimol infusion under gas anesthesia in the post-training period, consolidation of memory of the adaptation was markedly impaired, compared with the control mice which daily received bilateral floccular Ringer''s solution infusions under gas anesthesia or those which daily received only gas anesthesia. These results are consistent with the studies of the effects of inactivation of cerebellar cortex on the consolidation of motor memory of rabbit eyeblink conditioning , and suggest that the post-training cerebellar cortex activity play an important for the consolidation of motor memory of HOKR adaptation. [Copyright &y& Elsevier]