Mariko Miyata

Local Calcium Release in Dendritic Spines Required for Long-Term Synaptic Depression

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.

Corticotropin-Releasing Factor Plays a Permissive Role in Cerebellar Long-Term Depression

This study of rat cerebellar slices yielded two lines of evidence indicating that the corticotropin-releasing factor (CRF) found in climbing fibers (CFs) is critical for the induction of long-term depression (LTD) at the parallel fiber (PF) synapses of Purkinje cells (PCs) by their conjunctive activation with either stimulation of CFs or depolarization of PCs. First, LTD induction was effectively blocked by specific CRF receptor antagonists, alpha-helical CRF-(9-41) (alpha-h CRF) and astressin; and second, LTD was no longer observed in CF-deprived cerebella but was restored by CRF replenishment. The data obtained in this study suggest that these effects are mediated by protein kinase C (PKC) and not by Ca2+ signaling or cyclic GMP (cGMP) production.

Deficient long‐term synaptic depression in the rostral cerebellum correlated with impaired motor learning in phospholipase C β4 mutant mice

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

Roles of phospholipase Cbeta4 in synapse elimination and plasticity in developing and mature cerebellum.

The β isoforms of phospholipase C (PLCβs) are thought to mediate signals from metabotropic glutamate receptor subtype 1 (mGluR1) that is crucial for the modulation of synaptic transmission and plasticity. Among four PLCβ isoforms, PLCβ4 is one of the two major isoforms expressed in cerebellar Purkinje cells. The authors have studied the roles of PLCβ4 by analyzing PLCβ4 knock-out mice, which are viable, but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple climbing fiber innervation is clearly impaired in the rostral portion of the cerebellar vermis, where PLCβ4 mRNA is predominantly expressed in the wild-type mice. In the adult, long-term depression is deficient at parallel fiber to Purkinje cell synapses in the rostral cerebellum of the PLCβ4 knockout mice. The impairment of climbing fiber synapse elimination and the loss of long-term depression are similar to those seen in mice defective in mGluR1, Gαq, or protein kinase C. Thus, the authors’ results strongly suggest that PLCβ4 is part of a signaling pathway, including the mGluR1, Gαq and protein kinase C, which is crucial for both climbing fiber synapse elimination in the developing cerebellum and long-term depression induction in the mature cerebellum.

The synaptic targeting of mGluR1 by its carboxyl-terminal domain is crucial for cerebellar function.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca2+ release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.

Impaired Feedforward Inhibition of the Thalamocortical Projection in Epileptic Ca2+ Channel Mutant Mice, tottering

The tottering (tg) mice have a mutation in the CaV2.1 (P/Q-type) voltage-dependent Ca2+ channel α12.1 subunit gene. tg mice show not only cerebellar ataxia but also absence epilepsy, which begins at ∼3 weeks of age and persists throughout life. Similarities in EEG and sensitivity to antiepileptic drugs suggest that tg mice are a good model for human absence epilepsy. Although imbalance between excitatory and inhibitory activity in the thalamocortical network is thought to contribute to the pathogenesis of absence epilepsy, the effect of the mutation on thalamocortical synaptic responses remains unknown. Here we showed imbalanced impairment of inhibitory synaptic responses in tg mice using brain slice preparations. Somatosensory thalamocortical projection makes not only monosynaptic glutamatergic connections but also disynaptic GABAergic connections, which mediate feedforward inhibition, onto layer IV neurons. In tg mice, IPSC amplitudes recorded from layer IV pyramidal cells of the somatosensory cortex in response to thalamic stimulation became disproportionately reduced compared with EPSC amplitudes at later developmental stages (postnatal days 21–30). Similar results were obtained by local stimulation of layer IV pyramidal neurons. However, IPSC reduction was not seen in layer V pyramidal neurons of epileptic tg mice or in layer IV pyramidal neurons of younger tg mice before the onset of epilepsy (postnatal days 14–16). These results showed that the feedforward inhibition from the thalamus to layer IV neurons of the somatosensory cortex was severely impaired in tg mice and that the impairment of the inhibitory synaptic transmission was correlated to the onset of absence epilepsy.

A CaV2.1 calcium channel mutation rocker reduces the number of postsynaptic AMPA receptors in parallel fiber–Purkinje cell synapses

The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the CaV2.1 (P/Q‐type) Ca2+ channel α1 subunit, and show the mildest symptoms among the reported CaV2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)–PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF–PC synapses, however, was unexpectedly almost normal in terms of paired‐pulse facilitation, sensitivity to extracellular Ca2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze‐fracture replica labeling revealed that both the number and density of postsynaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF–PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice (∼ 1 month old). These lines of evidence suggest that even a moderate dysfunction of CaV2.1 Ca2+ channel can cause substantial changes in postsynaptic molecular composition of the PF–PC synapses and dendritic structure of PCs.

Different composition of glutamate receptors in corticothalamic and lemniscal synaptic responses and their roles in the firing responses of ventrobasal thalamic neurons in juvenile mice

Thalamic ventrobasal (VB) relay neurons receive information via two major types of glutamatergic synapses, that is, from the medial lemniscus (lemniscal synapses) and primary somatosensory cortex (corticothalamic synapses). These two synapses influence and coordinate firing responses of VB neurons, but their precise operational mechanisms are not yet well understood. In this study, we compared the composition of glutamate receptors and synaptic properties of corticothalamic and lemniscal synapses. We found that the relative contribution of NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs) to non‐NMDA receptor‐mediated EPSCs was significantly greater in corticothalamic synapses than in lemniscal synapses. Furthermore, NMDA receptor 2B‐containing NMDA receptor‐ and kainate receptor‐mediated currents were observed only in corticothalamic synapses, but not in lemniscal synapses. EPSCs in corticothalamic synapses displayed the postsynaptic summation in a frequency‐dependent manner, in which the summation of the NMDA receptor‐mediated component was largely involved. The summation of kainate receptor‐mediated currents also partially contributed to the postsynaptic summation in corticothalamic synapses. In contrast, the contribution of NMDA receptor‐mediated currents to the postsynaptic summation of lemniscal EPSCs was relatively minor. Furthermore, our results indicated that the prominent NMDA receptor‐mediated component in corticothalamic synapses was the key determinant for the late‐persistent firing of VB neurons in response to corticothalamic stimuli. In lemniscal synapses, in contrast, the onset‐transient firing in response to lemniscal stimuli was regulated mainly by AMPA receptors.

A Role for Myosin Va in Cerebellar Plasticity and Motor Learning: A Possible Mechanism Underlying Neurological Disorder in Myosin Va Disease

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

Electrophysiologic and functional evaluations of regenerated facial nerve defects with a tube containing dental pulp cells in rats.

Background: Dental pulp tissue contains Schwann and neural progenitor cells. Tissue-engineered nerve conduits with dental pulp cells promote facial nerve regeneration in rats. However, no nerve functional or electrophysiologic evaluations were performed. This study investigated the compound muscle action potential recordings and facial functional analysis of dental pulp cell regenerated nerve in rats. Methods: A silicone tube containing rat dental pulp cells in type I collagen gel was transplanted into a 7-mm gap of the buccal branch of the facial nerve in Lewis rats; the same defect was created in the marginal mandibular branch, which was ligatured. Compound muscle action potential recordings of vibrissal muscles and facial functional analysis with facial palsy score of the nerve were performed. Results: Tubulation with dental pulp cells showed significantly lower facial palsy scores than the autograft group between 3 and 10 weeks postoperatively. However, the dental pulp cell facial palsy scores showed no significant difference from those of autograft after 11 weeks. Amplitude and duration of compound muscle action potentials in the dental pulp cell group showed no significant difference from those of the intact and autograft groups, and there was no significant difference in the latency of compound muscle action potentials between the groups at 13 weeks postoperatively. However, the latency in the dental pulp cell group was prolonged more than that of the intact group. Conclusion: Tubulation with dental pulp cells could recover facial nerve defects functionally and electrophysiologically, and the recovery became comparable to that of nerve autografting in rats.