Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones.

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

Developmental stage-specific spontaneous activity contributes to callosal axon projections.

The developing neocortex exhibits spontaneous network activity with various synchrony levels, which has been implicated in the formation of cortical circuits. We previously reported that the development of callosal axon projections, one of the major long-range axonal projections in the brain, is activity dependent. However, what sort of activity and when activity is indispensable are not known. Here, using a genetic method to manipulate network activity in a stage-specific manner, we demonstrated that network activity contributes to callosal axon projections in the mouse visual cortex during a 'critical period': restoring neuronal activity during that period resumed the projections, whereas restoration after the period failed. Furthermore, in vivo Ca2+ imaging revealed that the projections could be established even without fully restoring highly synchronous activity. Overall, our findings suggest that spontaneous network activity is selectively required during a critical developmental time window for the formation of long-range axonal projections in the cortex.

Norepinephrine Facilitates Induction of Long-term Depression through ß-Adrenergic Receptor at Parallel Fiber-to-Purkinje Cell Synapses in the Flocculus.

The cerebellum is involved in motor learning, and long-term depression (LTD) at parallel fiber-to-Purkinje cell (PF-PC) synapses has been considered to be a primary cellular mechanism for motor learning. In addition, the contribution of norepinephrine (NE) to cerebellum-dependent learning paradigms has been reported. Thus, the roles of LTD and of NE in motor learning have been studied separately, and the relationship between the effects of NE and LTD remains unclear. Here, we examined effects of ß-adrenergic receptor (ß-AR) activity on the synaptic transmission and LTD at PF-PC synapses in the cerebellar flocculus. The flocculus regulates adaptation of oculomotor reflexes, and we previously reported the involvement of both LTD and ß-AR in adaptation of an oculomotor reflex. Here we found that specific agonists for ß-AR or NE did not directly change synaptic transmission, but lowered the threshold for LTD induction at PF-PC synapses in the flocculus. In addition, protein kinase A (PKA), which is activated downstream of ß-AR, facilitated the LTD induction. Altogether, these results suggest that NE facilitates LTD induction at PF-PC synapses in the flocculus by activating PKA through ß-AR.

Amyloid-ß oligomers suppress subunit-specific glutamate receptor increase during LTP.

INTRODUCTION: Amyloid-ß oligomers (AßOs) are assumed to impair the ability of learning and memory by suppressing the induction of synaptic plasticity, such as long-term potentiation (LTP) in the early stage of Alzheimer's disease. However, the direct molecular mechanism of how AßOs affect excitatory synaptic plasticity remains to be elucidated. METHODS: In order to study the effects of AßOs on LTP-associated changes of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptor (AMPAR) movement, we performed live-cell imaging of fluorescently labeled AMPAR subunit GluA1 or GluA2 with total internal reflection fluorescence microscopy. RESULTS: Incubation of cultured hippocampal neurons with AßOs for 1-2 days inhibited the increase in GluA1 number and GluA1 exocytosis frequency in both postsynaptic and extrasynaptic membranes during LTP. In contrast, AßOs did not inhibit the increase in GluA2 number or exocytosis frequency. DISCUSSION: These results suggest that AßOs primarily inhibit the increase in the number of GluA1 homomers and suppress hippocampal LTP expression.

Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface.

Regulation of exo- and endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) plays a critical role in the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) at excitatory central synapses. Enhanced AMPAR exocytosis or endocytosis has been suggested to contribute to LTP or LTD, respectively. However, several unsettled fundamental questions have remained about AMPAR exo- and endocytosis in the basal condition and during synaptic plasticity: (1) Does the size of each exo- or endocytosis event, and/or do the frequencies of these events change during LTP or LTD? If they change, what are the time courses of the respective changes? (2) Where does the exo- or endocytosis preferentially occur in each condition: inside or in the vicinity of postsynaptic membrane, or in the extrasynaptic membrane? (3) Do different types of AMPAR, such as GluA1 homo-tetramer, GluA1/2 hetero-tetramer and GluA2/3 hetero-tetramer, show distinct exo- and endocytosis changes? To address these questions, we developed new methods to observe individual events of AMPAR exo- or endocytosis with a high signal to noise (SN) ratio in a culture preparation using total internal reflection fluorescence microscopy (TIRFM). In these studies, hippocampal neurons were cultured on a neurexin (NRX)-coated glass coverslip, which induced formation of postsynaptic-like membrane (PSLM) directly on the glass surface. Then, a super-ecliptic pHluorin (SEP)-tagged AMPAR subunit such as GluA1 (GluA1-SEP) was expressed in neurons and its fluorescence changes during LTP induced by high frequency electrical field stimulation were observed with TIRFM, which showed different time courses of exocytosis changes of GluA1-, GluA2-, or GluA3-SEP in and around PSLM. In addition, a new method to detect individual endocytosis events of AMPAR was developed by combining TIFRM observation of GluA-SEP around PSLM with a rapid extracellular pH exchange method using a U-tube. Recent results on exo- and endocytosis changes of GluA-SEP during N-methyl-D-aspartate (NMDA)-induced LTD suggested that suppression of AMPAR exocytosis rather than enhancement of AMPAR endocytosis primarily contributes to LTD expression, although the NMDA application transiently enhances clathrin-dependent endocytosis of GluA1-containing AMPAR.

Purkinje Neurons: Development, Morphology, and Function.

Cerebellar Purkinje neurons are arguably some of the most conspicuous neurons in the vertebrate central nervous system. They have characteristic planar fan-shaped dendrites which branch extensively and fill spaces almost completely with little overlap. This dendritic morphology is well suited to receiving a single or a few excitatory synaptic inputs from each of more than 100,000 parallel fibers which run orthogonally to Purkinje cell dendritic trees. In contrast, another type of excitatory input to a Purkinje neuron is provided by a single climbing fiber, which forms some hundreds to thousands of synapses with a Purkinje neuron. This striking contrast between the two types of synaptic inputs to a Purkinje neuron has attracted many neuroscientists. It is also to be noted that Purkinje neurons are the sole neurons sending outputs from the cerebellar cortex. In other words, all computational results within the cortex are transmitted by Purkinje cell axons, which inhibit neurons in the cerebellar or vestibular nucleus. Notably, Purkinje neurons show several forms of synaptic plasticity. Among them, long-term depression (LTD) at parallel fiber synapses has been regarded as a putatively essential mechanism for cerebellum-dependent learning. In this special issue on Purkinje neurons, you will find informative reviews and original papers on the development, characteristics and functions of Purkinje neurons, or related themes contributed by outstanding researchers.

Author Correction: Synaptic N6-methyladenosine (m6A) epitranscriptome reveals functional partitioning of localized transcripts.

In the version of this article initially published, a Supplementary Fig. 6f was cited in the last paragraph of the Results. No such panel exists; the citation has been deleted. The error has been corrected in the HTML and PDF versions of the article.

Regulation and Interaction of Multiple Types of Synaptic Plasticity in a Purkinje Neuron and Their Contribution to Motor Learning.

There are multiple types of plasticity at both excitatory glutamatergic and inhibitory GABAergic synapses onto a cerebellar Purkinje neuron (PN). At parallel fiber to PN synapses, long-term depression (LTD) and long-term potentiation (LTP) occur, while at molecular layer interneuron to PN synapses, a type of LTP called rebound potentiation (RP) takes place. LTD, LTP, and RP seem to contribute to motor learning. However, each type of synaptic plasticity might play a different role in various motor learning paradigms. In addition, defects in one type of synaptic plasticity could be compensated by other forms of synaptic plasticity, which might conceal the contribution of a particular type of synaptic plasticity to motor learning. The threshold stimulation for inducing each type of synaptic plasticity and the induction conditions are different for different plasticity mechanisms, and they change depending on the state of an animal. Facilitation and/or saturation of synaptic plasticity occur after certain behavioral experiences or in some transgenic mice. Thus, the regulation and roles of synaptic plasticity are complicated. Toward a comprehensive understanding of the respective roles of each type of synaptic plasticity and their possible interactions during motor learning processes, I summarize induction conditions, modulations, interactions, and saturation of synaptic plasticity and discuss how multiple types of synaptic plasticity in a PN might work together in motor learning processes.

Synaptic N6-methyladenosine (m6A) epitranscriptome reveals functional partitioning of localized transcripts.

A localized transcriptome at the synapse facilitates synapse-, stimulus- and transcript-specific local protein synthesis in response to neuronal activity. While enzyme-mediated mRNA modifications are known to regulate cellular mRNA turnover, the role of these modifications in regulating synaptic RNA has not been studied. We established low-input m6A-sequencing of synaptosomal RNA to determine the chemically modified local transcriptome in healthy adult mouse forebrains and identified 4,469 selectively enriched m6A sites in 2,921 genes as the synaptic m6A epitranscriptome (SME). The SME is functionally enriched in synthesis and modulation of tripartite synapses and in pathways implicated in neurodevelopmental and neuropsychiatric diseases. Interrupting m6A-mediated regulation via knockdown of readers in hippocampal neurons altered expression of SME member Apc, resulting in synaptic dysfunction including immature spine morphology and dampened excitatory synaptic transmission concomitant with decreased clusters of postsynaptic density-95 (PSD-95) and decreased surface expression of AMPA receptor subunit GluA1. Our findings indicate that chemical modifications of synaptic mRNAs critically contribute to synaptic function.

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17-0.19 µm2/s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Ulk2 controls cortical excitatory-inhibitory balance via autophagic regulation of p62 and GABAA receptor trafficking in pyramidal neurons.

Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.

Suppression of AMPA Receptor Exocytosis Contributes to Hippocampal LTD.

The decrease in number of AMPA-type glutamate receptor (AMPAR) at excitatory synapses causes LTD, a cellular basis of learning and memory. The number of postsynaptic AMPARs is regulated by the balance of exocytosis and endocytosis, and enhanced endocytosis of AMPAR has been suggested to underlie the LTD expression. However, it remains unclear how endocytosis and exocytosis of AMPAR change during LTD. In this study, we addressed this question by analyzing exocytosis and endocytosis of AMPAR by imaging super-ecliptic pHlorin (SEP)-tagged AMPAR around postsynaptic structure formed directly on the glass surface in the hippocampal culture prepared from rat embryos of both sexes. Contrary to a prevailing view on the LTD expression by endocytosis enhancement, the LTD induction by NMDA application only transiently enhanced endocytosis of SEP-tagged GluA1 subunits of AMPAR, which was counteracted by simultaneous augmentation of exocytosis. As a result, soon after the start of the LTD induction (∼1 min), the surface AMPAR did not markedly decrease. Thereafter, the surface GluA1-SEP gradually decreased (2-5 min) and kept at a low level until the end of observation (>30 min). Surprisingly, this gradual and sustained decrease of surface AMPAR was accompanied not by the enhanced endocytic events of GluA1, but by the suppression of exocytosis. Together, our data highlight an unprecedented mechanism for the LTD expression by attenuation of exocytosis of AMPAR, but not by enhanced endocytosis, together with a reduction of postsynaptic AMPAR scaffolding protein PSD95.SIGNIFICANCE STATEMENT It has been generally assumed that LTD is expressed by enhancement of AMPAR endocytosis. Previous studies reported that endocytosis-related protein was involved in LTD and that significant amount of cell-surface AMPAR moved into intracellular compartments during LTD. Here, we report changes of cell-surface amount of AMPAR, and where and when individual exocytosis and endocytosis occurred during LTD. Cell-surface AMPAR gradually decreased in synchrony with suppression of exocytosis but not with enhancement of endocytosis. These results suggest that the decrease of cell-surface AMPAR amount during LTD was caused not by enhancement of endocytosis but rather by suppression of exocytosis, which revises current understanding of the expression mechanism of LTD.

Occurrence of long-term depression in the cerebellar flocculus during adaptation of optokinetic response.

Long-term depression (LTD) at parallel fiber (PF) to Purkinje cell (PC) synapses has been considered as a main cellular mechanism for motor learning. However, the necessity of LTD for motor learning was challenged by demonstration of normal motor learning in the LTD-defective animals. Here, we addressed possible involvement of LTD in motor learning by examining whether LTD occurs during motor learning in the wild-type mice. As a model of motor learning, adaptation of optokinetic response (OKR) was used. OKR is a type of reflex eye movement to suppress blur of visual image during animal motion. OKR shows adaptive change during continuous optokinetic stimulation, which is regulated by the cerebellar flocculus. After OKR adaptation, amplitudes of quantal excitatory postsynaptic currents at PF-PC synapses were decreased, and induction of LTD was suppressed in the flocculus. These results suggest that LTD occurs at PF-PC synapses during OKR adaptation.

Differential regulations of vestibulo-ocular reflex and optokinetic response by ß- and α2-adrenergic receptors in the cerebellar flocculus.

Norepinephrine modulates synaptic plasticity in various brain regions and is implicated in memory formation, consolidation and retrieval. The cerebellum is involved in motor learning, and adaptations of the vestibulo-ocular reflex (VOR) and optokinetic response (OKR) have been studied as models of cerebellum-dependent motor learning. Previous studies showed the involvement of adrenergic systems in the regulation of VOR, OKR and cerebellar synaptic functions. Here, we show differential contributions of ß- and α2-adrenergic receptors in the mouse cerebellar flocculus to VOR and OKR control. Effects of application of ß- or α2-adrenergic agonist or antagonist into the flocculus suggest that the ß-adrenergic receptor activity maintains the VOR gain at high levels and contributes to adaptation of OKR, and that α2-adrenergic receptor counteracts the ß-receptor activity in VOR and OKR control. We also examined effects of norepinephrine application, and the results suggest that norepinephrine regulates VOR and OKR through ß-adrenergic receptor at low concentrations and through α2-receptor at high concentrations.

Detection and characterization of individual endocytosis of AMPA-type glutamate receptor around postsynaptic membrane.

Synaptic plasticity such as long-term depression (LTD) has been regarded as a cellular mechanism of learning and memory. LTD is expressed by the decrease in number of postsynaptic AMPA-type receptor (AMPAR) at glutamatergic synapses. Although endocytosis is known to play an essential role in the decrease in AMPAR on postsynaptic membrane, the difficulty to detect individual endocytic events hampered clarification of AMPAR dynamics around synapses. Previously, we developed a method to induce formation of postsynaptic-like membrane (PSLM) on the glass surface and observed pHluorin-tagged AMPAR around PSLM with total internal reflection fluorescence microscopy. By this method, individual exocytosis of AMPAR-pHluorin was recorded in both PSLM and non-PSLM. In other studies, endocytic vesicles containing pHluorin-tagged receptors were visualized by changing extracellular pH. Here, we have combined PSLM formation method and rapid pH change method, and detected individual endocytic events of AMPAR around PSLM with high spatial and temporal resolutions. Endocytic events of AMPAR were characterized by comparison with those of transferrin receptor. Constitutive endocytosis of AMPAR was not dependent on clathrin and dynamin in contrast to that of transferrin receptor. However, AMPAR endocytosis triggered by LTD-inducing stimulation was clathrin- and dynamin-dependent.

Intracellular Ca(2+) thresholds for induction of excitatory long-term depression and inhibitory long-term potentiation in a cerebellar Purkinje neuron.

Synaptic plasticity in the cerebellar cortex contributes to motor learning. In particular, long-term depression at excitatory parallel fiber - Purkinje neuron synapses has been intensively studied as a primary cellular mechanism for motor learning. Recent studies showed that synaptic plasticity other than long-term depression such as long-term potentiation at inhibitory interneuron - Purkinje neuron synapses called rebound potentiation is also involved in motor learning. It was suggested that long-term depression and rebound potentiation might synergistically support motor learning. Here, we have examined induction conditions of long-term depression and rebound potentiation in cultured rat Purkinje neurons, and found that both of them were induced simultaneously by certain patterns of depolarization of a Purkinje neuron. Further, we found that long-term depression was induced by shorter depolarizing pulses causing a smaller intracellular Ca(2+) increase than rebound potentiation. These results support an idea that long-term depression and rebound potentiation synergistically contribute to motor learning, and suggest that long-term depression may play a primary role in wider variety of motor learning paradigms than rebound potentiation.

LTD, RP, and Motor Learning.

Long-term depression (LTD) at excitatory synapses between parallel fibers and a Purkinje cell has been regarded as a critical cellular mechanism for motor learning. However, it was demonstrated that normal motor learning occurs under LTD suppression, suggesting that cerebellar plasticity mechanisms other than LTD also contribute to motor learning. One candidate for such plasticity is rebound potentiation (RP), which is long-term potentiation at inhibitory synapses between a stellate cell and a Purkinje cell. Both LTD and RP are induced by the increase in postsynaptic Ca(2+) concentration, and work to suppress the activity of a Purkinje cell. Thus, LTD and RP might work synergistically, and one might compensate defects of the other. RP induction is dependent on the interaction between GABAA receptor and GABAA receptor binding protein (GABARAP). Transgenic mice expressing a peptide which inhibits binding of GABARAP and GABAA receptor only in Purkinje cells show defects in both RP and adaptation of vestibulo-ocular reflex (VOR), a motor learning paradigm. However, another example of motor learning, adaptation of optokinetic response (OKR), is normal in the transgenic mice. Both VOR and OKR are reflex eye movements suppressing the slip of visual image on the retina during head movement. Previously, we reported that delphilin knockout mice show facilitated LTD induction and enhanced OKR adaptation, but we recently found that VOR adaptation was not enhanced in the knockout mice. These results together suggest that animals might use LTD and RP differently depending on motor learning tasks.

Control of Spontaneous Ca2+ Transients Is Critical for Neuronal Maturation in the Developing Neocortex.

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

Opposite regulation of inhibitory synaptic plasticity by α and ß subunits of Ca(2+)/calmodulin-dependent protein kinase II.

Induction of several forms of synaptic plasticity, a cellular basis for learning and memory, depends on the activation of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII). CaMKII acts as a holoenzyme consisting of α and ß subunits (α- and ßCaMKII). However, it remains elusive how the subunit composition of a CaMKII holoenzyme affects its activation and hence synaptic plasticity. We addressed this issue by focusing on long-term potentiation (LTP) at inhibitory synapses on cerebellar Purkinje neurons (PNs) (called rebound potentiation, RP). The contribution of each subunit to RP was examined by selective knock-down or overexpression of that subunit. Electrophysiological recording from a rat cultured PN demonstrated that ßCaMKII is essential for RP induction, whereas αCaMKII suppresses it. Thus, RP was negatively regulated due to the greater relative abundance of αCaMKII compared to ßCaMKII, suggesting a critical role of CaMKII subunit composition in RP. The higher affinity of ßCaMKII to Ca(2+)/CaM compared with αCaMKII was responsible for the predominant role in RP induction. Live-cell imaging of CaMKII activity based on the Förster resonance energy transfer (FRET) technique revealed that ßCaMKII enrichment enhances the total CaMKII activation upon a transient conditioning depolarization. Taken together, these findings clarified that α- and ßCaMKII oppositely regulate CaMKII activation, controlling the induction of inhibitory synaptic plasticity in a PN, which might contribute to the adaptive information processing of the cerebellar cortex.

Around LTD hypothesis in motor learning.

Long-term depression (LTD) at parallel fiber-Purkinje neuron synapses has been regarded as a primary cellular mechanism for motor learning. However, this hypothesis has been challenged. Demonstration of normal motor learning under LTD-suppressed conditions suggested that motor learning can occur without LTD. Synaptic plasticity mechanisms other than LTD have been found at various synapses in the cerebellum. Animals may achieve motor learning using several types of synaptic plasticity in the cerebellum including LTD.