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DOI： 10.1038/s41380-018-0295-y

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Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.

DOI： 10.1038/s41380-018-0074-9

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

Outputs from the cerebellar nuclei (CN) are important for generating and controlling movement. The activity of CN neurons is controlled not only by excitatory inputs from mossy and climbing fibers and by γ-aminobutyric acid (GABA)-based inhibitory transmission from Purkinje cells in the cerebellar cortex but is also modulated by inputs from other brain regions, including serotonergic fibers that originate in the dorsal raphe nuclei. We examined the modulatory effects of serotonin (5-HT) on GABAergic synapses during development, using rat cerebellar slices. As previously reported, 5-HT presynaptically decreased the amplitudes of stimulation-evoked inhibitory postsynaptic currents (IPSCs) in CN neurons, with this effect being stronger in slices from younger animals (postnatal days [P] 11–13) than in slices from older animals (P19–21). GABA release probabilities accordingly exhibited significant decreases from P11–13 to P19–21. Although there was a strong correlation between the GABA release probability and the magnitude of 5-HT-induced inhibition, manipulating the release probability by changing extracellular Ca2+ concentrations failed to control the extent of 5-HT-induced inhibition. We also found that the IPSCs exhibited slower kinetics at P11–13 than at P19–21. Pharmacological and molecular biological tests revealed that IPSC kinetics were largely determined by the prevalence of α1 subunits within GABAA receptors. In summary, pre- and postsynaptic developmental changes in serotonergic modulation and GABAergic synaptic transmission occur during the second to third postnatal weeks and may significantly contribute to the formation of normal adult cerebellar function.

DOI： 10.1007/s12311-018-0922-9

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Previous studies have shown that phenotypic modulation of smooth muscle cells (SMCs) plays a pivotal role in human diseases. However, the molecular mechanisms underlying the reversible differentiation of SMCs remain elusive particularly because cultured SMCs that reproducibly exhibit bidirectional phenotypic modulation have not been established. Here we established an immortalized human bladder SMC line designated as hBS11. Under differentiation-inducing conditions, hBS11 cells underwent smooth muscle differentiation accompanied by the robust expression of smooth muscle differentiation markers and isoform-dependent reorganization of actin bundles. The cholinergic receptor agonist carbachol increased intracellular calcium in differentiated hBS11 cells in an acetylcholine muscarinic receptor-dependent manner. Differentiated hBS11 cells displayed contractile properties depending on the elevation in the levels of intracellular calcium. Depolarization of membrane potential triggered inward sodium current in differentiated hBS11 cells. However, differentiated hBS11 cells lost the differentiated phenotype and resumed mitosis when re-fed with growth medium. Our study provides direct evidence pertaining to the human bladder SMCs being able to retain the capacity of reversible differentiation and that the reorganization of actin bundles is involved in the reinstatement of contractility. Moreover, we have established a human SMC line retaining high proliferating potential without compromising differentiation potential.

DOI： 10.1371/journal.pone.0186584

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

miR-17-92 is a microRNA cluster with six distinct members. Here, we show that the miR-17-92 cluster and its individual members modulate chronic neuropathic pain. All cluster members are persistently upregulated in primary sensory neurons after nerve injury. Overexpression of miR-18a, miR-19a, miR-19b and miR-92a cluster members elicits mechanical allodynia in rats, while their blockade alleviates mechanical allodynia in a rat model of neuropathic pain. Plausible targets for the miR-17-92 cluster include genes encoding numerous voltage-gated potassium channels and their modulatory subunits. Single-cell analysis reveals extensive co-expression of miR-17-92 cluster and its predicted targets in primary sensory neurons. miR-17-92 downregulates the expression of potassium channels, and reduced outward potassium currents, in particular A-type currents. Combined application of potassium channel modulators synergistically alleviates mechanical allodynia induced by nerve injury or miR-17-92 overexpression. miR-17-92 cluster appears to cooperatively regulate the function of multiple voltage-gated potassium channel subunits, perpetuating mechanical allodynia.

DOI： 10.1038/ncomms16079

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Serotonin is a critical modulator of cortical function, and its metabolism is defective in autism spectrum disorder (ASD) brain. How serotonin metabolism regulates cortical physiology and contributes to the pathological and behavioral symptoms of ASD remains unknown. We show that normal serotonin levels are essential for the maintenance of neocortical excitation/inhibition balance, correct sensory stimulus tuning, and social behavior. Conversely, low serotonin levels in 15q dup mice (a model for ASD with the human 15q11-13 duplication) result in impairment of the same phenotypes. Restoration of normal serotonin levels in 15q dup mice revealed the reversibility of a subset of ASD-related symptoms in the adult. These findings suggest that serotonin may have therapeutic potential for discrete ASD symptoms.

DOI： 10.1126/sciadv.1603001

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Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABA(A) receptors in PCs of rat cerebellar cortex during depolarization-induced "rebound potentiation (RP)" and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABA(A) receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABA(A) receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABA(A) receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y11 purinoceptor. Thus, our findings reveal that postsynaptic GABA(A) receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.

DOI： 10.1371/journal.pone.0150636

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.

DOI： 10.1128/MCB.01339-14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct GAD autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal GAD antibodies. We found that GAD autoantibodies present in patients with stiff person syndrome (n = 7) and cerebellar ataxia (n = 15) recognized an epitope distinct from that recognized by GAD autoantibodies present in patients with type 1 diabetes mellitus p = 10) or limbic encephalitis (n = 4). We demonstrated that the administration of a monoclonal GAD antibody representing this epitope specificity; (1) disrupted in vitro the association of GAD with y-Aminobutyric acid containing synaptic vesicles: (2) depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect; (3) significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task; (4) markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired pulse stimulation paradigm; and (5) induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of GAD by its autoantibodies in the pathogenesis of stiff person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such GAD antibodies could be envisioned.

DOI： 10.3389/fnbeh.2015.00078

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Publishing type：Research paper (scientific journal)   Publisher：Wiley Online Library  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Neuronal damage in the somatosensory system causes intractable chronic neuropathic pain. Plastic changes in sensory neuron excitability are considered the cellular basis of persistent pain. Non-coding microRNAs modulate specific gene translation to impact on diverse cellular functions and their dysregulation causes various diseases. However, their significance in adult neuronal functions and disorders is still poorly understood. Here, we show that miR-7a is a key functional RNA sustaining the late phase of neuropathic pain through regulation of neuronal excitability in rats. In the late phase of neuropathic pain, microarray analysis identified miR-7a as the most robustly decreased microRNA in the injured dorsal root ganglion. Moreover, local induction of miR-7a, using an adeno-associated virus vector, in sensory neurons of injured dorsal root ganglion, suppressed established neuropathic pain. In contrast, miR-7a overexpression had no effect on acute physiological or inflammatory pain. Furthermore, miR-7a downregulation was sufficient to cause pain-related behaviours in intact rats. miR-7a targeted the beta 2 subunit of the voltage-gated sodium channel, and decreased miR-7a associated with neuropathic pain caused increased beta 2 subunit protein expression, independent of messenger RNA levels. Consistently, miR-7a overexpression in primary sensory neurons of injured dorsal root ganglion suppressed increased beta 2 subunit expression and normalized long-lasting hyperexcitability of nociceptive neurons. These findings demonstrate miR-7a downregulation is causally involved in maintenance of neuropathic pain through regulation of neuronal excitability, and miR-7a replenishment offers a novel therapeutic strategy specific for chronic neuropathic pain.

DOI： 10.1093/brain/awt191

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Am Physiological Soc  

DOI： 10.1002/phy2.61

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67-green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT1A or 5-HT2A/2C receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches. © 2012 The Author(s).

DOI： 10.1007/s12576-012-0250-7

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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.

DOI： 10.1371/journal.pone.0029663

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The deep cerebellar nuclei (DCN) are the terminal components of the cerebellar circuitry and constitute its primary output structure. Their activity is important for certain forms of motor learning as well as generation and control of movement. DCN neurons receive glutamatergic excitatory inputs from the pontine nuclei via mossy fibres (MFs) and concomitantly receive inputs from 5-HT-containing neurons of the raphe nuclei. We aimed to explore the roles of 5-HT at MF-DCN synapses by using cerebellar slices from 11 to 15-day-old rats. Bath application of 5-HT reversibly decreased the amplitude of stimulation-evoked excitatory postsynaptic currents (eEPSCs) via the activation of 5-HT(1B) receptors at the presynaptic terminals of the MFs. Burst stimulation of the MFs elicited long-term depression (LTD) at the MF-DCN synapses that require activation of the group I metabotropic glutamate receptor (mGluR). In the presence of 5-HT, the extent of burst-induced LTD of MF EPSCs was significantly reduced. Application of 5-HT also decreased the amplitude of mGluR-dependent slow EPSCs evoked by similar burst stimulation. Furthermore, (S)-3,5-dihydroxyphenylglycine (DHPG), a group I mGluR agonist, induced chemical LTD of MF EPSCs, and 5-HT had no significant effect on this LTD. Taken together, the results suggest that 5-HT not only has transitory inhibitory effects on MF EPSCs but also plays a role in regulating the long-term synaptic efficacy. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2010.10.037

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Saitow F, Murano M, Suzuki H. Modulatory effects of serotonin on GABAergic synaptic transmission and membrane properties in the deep cerebellar nuclei. J Neurophysiol 101: 1361-1374, 2009. First published January 14, 2009; doi:10.1152/jn.90750.2008. Cerebellar outputs from the deep cerebellar nuclei (DCN) are critical for generating and controlling movement. DCN neuronal activity is primarily controlled by GABAergic inhibitory transmission by Purkinje cells in the cerebellar cortex and is also modulated by nerve inputs originating from other brain regions within and outside the cerebellum. In this study, we examined the modulatory effects of 5-HT on GABAergic synapses in the DCN. 5-HT decreased the amplitude of stimulation-evoked inhibitory postsynaptic currents (eIPSCs) in DCN neurons, and this effect was abolished by a 5-HT(1B) antagonist, SB 224289. The decrease in IPSC amplitude was associated with an increased paired-pulse ratio of the IPSC. 5-HT also decreased the frequency of miniature IPSCs without altering the amplitude. These data suggest that 5-HT presynaptically inhibited GABA release. Furthermore, 5-HT elicited a slow inward current in DCN neurons. Pharmacological studies showed that 5-HT activated the 5-HT(5) receptor, which is positively coupled to G protein and elicited the slow inward current through enhancement of hyperpolarization-activated cation channel activation. Finally, we examined the effects of 5-HT on the spike generation that accompanies repetitive stimulation of inhibitory synapses. 5-HT increased the spontaneous firing rate in DCN neurons caused by depolarization. Increase in the 5-HT-induced tonic firing relatively decreased the contrast difference from the rebound depolarization-induced firing. However, the inhibitory transmission-induced silencing of DCN firing remained during the conditioning stimulus. These results suggest that 5-HT plays a regulatory role in spike generation and contributes to the gain control of inhibitory GABAergic synapses in DCN neurons.

DOI： 10.1152/jn.90750.2008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

1 Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK-1 and NK-3 tachykinin receptors ( referred to as rmNK-1 and rmNK-3) from the rhesus monkey ( Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS.
2 The deduced rmNK-1 amino-acid sequence differed by only two amino acids from the human NK-1 (hNK-1). The deduced rmNK-3 amino-acid sequence was two amino acids shorter than human NK-3 (hNK-3), with a seven-amino-acid difference in sequence.
3 Ligand binding studies revealed that the affinity of rmNK-1 to substance P (SP) was comparable to that of hNK-1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK-1 and hNK-1. Affinity of rmNK-3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK-3. Ca2+ imaging showed that activations of both rmNK-1 and rmNK-3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors.
4 The amounts of rmNK-1 and rmNK-3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK-1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK-1 mRNA in the brain, with less rmNK-3 mRNA found in the cortex compared to rmNK-1 mRNA. In the monkey hippocampus and amygdala, rmNK-1 mRNA was present at markedly higher concentrations than rmNK-3 mRNA.
5 The present results provide an insight into the distinct physiological nature and significance of the NK-1 and NK-3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype-specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.

DOI： 10.1038/sj.bjp.0706561

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Properly regulated interactions among excitatory and inhibitory synapses are critical for brain function. Compared to excitatory synapses, much less is known about the gain control mechanisms at inhibitory synapses. Herein we report a mechanism of noradrenergic long-term potentiation (LTP) at inhibitory synapses following presynaptic beta-adrenoceptor activation. Stimulation of beta-adrenoceptors elicited LTP of GABA release from terminals of cerebellar interneurones. This action was dependent on the cAMP/protein kinase A signalling cascade and independent of the beta-adrenoceptor-mediated acceleration of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel. Furthermore, the beta-adrenoceptor- and protein kinase A-mediated UP was triggered by enhancement of the Ca2+ sensitivity of the release machinery and increase in the readily releasable pool. beta-Adrenoceptor activation also accelerated the recruitment of GABA into the releasable pool and enhanced synchronous and asynchronous release of GABA from the presynaptic terminal. Thus, the up-regulation of GABA release machinery mediated by noradrenaline and beta-adrenoceptor activation provides a likely mechanism of feedforward inhibition of the cerebellar output neurone Purkinje cell, leading to a profound effect on motor control and learning associated with the cerebellum.

DOI： 10.1113/jphysiol.2005.084384

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Cerebellar Purkinje cells ( PCs) receive GABAergic input that undergoes powerful retrograde modulation by presynaptic cannabinoid and glutamate receptors. Here we examine a distinct modulatory mechanism at these synapses, which does not require postsynaptic depolarization and acts via presynaptic AMPA receptors. We find that this mechanism operates mainly in the somatic vicinity of PCs in which large boutons of basket cell axons form synapses on the PC soma. We use fast confocal microscopy and detailed kinetic modeling to estimate that, in these boutons, an action potential opens 100 - 200 Ca(2+) channels, eliciting a brief 3 - 5 mu M transient, followed by a longer-term, 15 - 30 nM rise of free Ca(2+) ( above the resting level of similar to 100 nM). Brief activation of local AMPA receptors suppresses Ca(2+) entry ( probably by silencing 20 - 40 P/Q- type channels) in a subgroup of terminals that tend to show a higher dynamic range of Ca(2+) signaling. The results provide the first quantitative description of presynaptic Ca(2+) kinetics and its modulation by AMPA receptor activation ( most likely via a glutamate spillover-mediated mechanism) at identified GABAergic synapses.

DOI： 10.1523/JNEUROSCI.0338-05.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Cerebellar GABAergic inhibitory transmission is under heterosynaptic control mediated by diverse chemical messengers. Here, we investigated roles of metabotropic P2Y purinoceptors (P2YRs) on GABAergic synapses between cerebellar interneurons and Purkinje cells (PCs). Activation of P2Y purinoceptors by two selective agonists, ADP and 2-methylthio-ADP (2MeSADP), elicited two distinct forms of synaptic plasticity of GABAergic transmission in the cerebellar cortex. First, the two agonists induced long-lasting enhancement of stimulation-evoked GABAergic IPSCs as well as GABA(A) receptor currents in PCs. This effect was completely abolished by intracellular infusion of the Ca2+- chelating agent BAPTA. Measurements of intracellular Ca2+ ([Ca2+](i)) dynamics showed that puff application of 2MeSADP produced an increase in [Ca2+](i) of PCs and that this increase persisted in an external Ca2+ -deficient medium. These results suggest that P2Y activation postsynaptically elicits long-term enhancement of GABA(A) receptor sensitivity of PCs through a G(q)-mediated increase in [Ca2+](i). The other action of P2YR agonists on cerebellar GABAergic synapses was that they produced a short-term increase in the frequency and the amplitude of spontaneous GABA(A) receptor-mediated IPSCs in PCs in a manner sensitive to a P2Y(1)R antagonist, N-6-methyl 2'-deoxyadenosine 3', 5'-bisphosphate. This action appeared to be attributable to an excitability increase in presynaptic GABAergic interneurons, because ADP excited all Lugaro cells examined and some of interneurons in the molecular layer. These results suggest that activation of cerebellar P2Y purinoceptors leads to modulation of GABAergic transmission in different spatial and temporal domains, namely short-term and long-term plasticity through presynaptic and postsynaptic mechanisms at interneuron-->PC inhibitory synapses in the rat cerebellar cortex.

DOI： 10.1523/JNEUROSCI.4254-04.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

A major subtype of glutamate receptors, AMPA receptors (AMPARs), are generally thought to mediate excitation at mammalian central synapses via the ionotropic action of ligand-gated channel opening. It has recently emerged, however, that synaptic activation of AMPARs by glutamate released from the climbing fibre input elicits not only postsynaptic excitation but also presynaptic inhibition of GABAergic transmission onto Purkinje cells in the cerebellar cortex. Although presynaptic inhibition is critical for information processing at central synapses, the molecular mechanisms by which AMPARs take part in such actions are not known. This study therefore aimed at further examining the properties of AMPAR-mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum. Our data provide evidence that the climbing fibre-induced inhibition of GABA release from interneurons depends on AMPAR-mediated activation of GTP-binding proteins coupled with down-regulation of presynaptic voltage-dependent Ca2+ channels. A G(i/o)-protein inhibitor, N-ethylmaleimide, selectively abolished the AMPAR-mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR-mediated excitatory actions on Purkinje cells. Furthermore, both G(i/o)-coupled receptor agonists, baclofen and DCG-IV, and the P/Q-type calcium channel blocker omega-agatoxin IVA markedly occluded the AMPAR-mediated inhibition of GABAergic transmission. Conversely, AMPAR activation inhibited action potential-triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons. By suppressing the inhibitory inputs to Purkinje cells, the AMPAR-mediated presynaptic inhibition could thus provide a feed-forward mechanism for the information flow from the cerebellar cortex.

DOI： 10.1111/j.1460-9568.2004.03347.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：VSP BV  

Noradrenaline (NA) and serotonin (5-HT) released by electrical stimulation into the rat cerebellar cortex from afferent input terminals have been shown to elicit long-term facilitation of inhibitory GABAergic transmission between interneurons, basket cells (BCs), and Purkinje cells (PCs). This study aimed to further examine receptor mechanisms underlying the noradrenergic facilitation. Using cerebellar slices cut from neonatal rats and patch-damp recordings, we explored the actions of adrenoceptor agonists and antagonists on GABAergic synapses. GABA-mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation and recorded from PCs. Application of NA or isoproterenol (ISP), a beta -receptor agonist increased the amplitude of IPSCs and the frequency of miniature IPSCs without affecting postsynaptic GABA receptor sensitivity and mean amplitude of miniature IPSCs. The enhancement by NA of IPSCs was suppressed by a beta (2)-adrenoceptor antagonist, ICI118,551, but not try a beta (1)-adrenoceptor antagonist, CGP20712A, suggesting that the beta (2)-adrenoceptors on BCs mediate the noradrenergic facilitation of GABAergic transmission Then we performed double recordings from BCs and PCs, which showed that the beta -agonist ISP increased the frequencies of the spontaneous spikes in BCs and the spike-triggered IPSCs in PCs. Forskolin mimicked the actions of beta -agonist in enhancing BC spiking and spike-triggered IPSCs in PCs. Furthermore voltage-clamp experiments showed that BCs exhibit profound activity of a hyperpolarization-activated cation channel current, I-h and that ISP and forskolin enhanced persistent activation of I-h channel. NA and ISP induced BC depolarization which was abolished by the I-h inhibitor ZD7288. Taken together, our data suggest that beta -adrenoceptor-mediated acceleration of I-h, in BCs is involved, at least in part, in noradrenergic facilitation of GABAergic transmission at BC-PC inhibitory synapses.

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In the preceding paper, we showed that norepinephrine (NE) enhances the spontaneous spike firings in cerebellar interneurons, basket cells (BCs), resulting in an increase in the frequency of BC-spike-triggered inhibitory postsynaptic currents (IPSCs) in Purkinje cells (PCs), and that the effects of NE on GABAergic BCs are mediated by beta(2)-adrenergic receptors. This study aimed to further examine the ionic mechanism underlying the beta-adrenoceptor-mediated facilitation of GABAergic transmission at the BC-PC synapses. Using cerebellar slices obtained from 15- to 21-day-old rats and whole cell recordings, we investigated ionic currents in the BCs and the effects of the beta-agonist isoproterenol (ISP) as well as forskolin on the BC excitability. Hyperpolarizing voltage steps from a holding potential of -50 mV elicited a hyperpolarization-activated inward current, I-h, in the BC. This current exhibited voltage-dependent activation that was accelerated by strong hyperpolarization, displaying two time constants, 84 +/- 6 and 310 +/- 40 ms, at -100 mV, and was inhibited by 20 mu M ZD7288. ISP and forskolin, both at 20 mu M, enhanced I-h by shifting the activation curve by 5.9 and 9.3 mV toward positive voltages, respectively. Under the current-clamp mode, ISP produced a depolarization of 7 +/- 3 mV in BCs and reduced their input resistance to 74 +/- 6%. ISP and a cAMP analogue, Rp-cAMP-S, increased the frequency of spontaneous spikes recorded from BCs using the cell-attached mode. The I-h inhibitor ZD7288 decreased the BC spike frequency and abolished the ISP-induced increase in spike discharges. The results suggest that NE depolarizes the BCs through beta-adrenoceptor-mediated cAMP formation linking it to activation of I-h, which is, at least in part, involved in noradrenergic afferent-mediated facilitation of GABAergic synaptic activity at BC-PC connections in the rat cerebellum.

DOI： 10.1152/jn.2000.84.4.2026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Norepinephrine (NE) has been shown to elicit long-term facilitation of GABAergic transmission to rat cerebellar Purkinje cells (PCs) through beta-adrenergic receptor activation. To further examine the locus and adrenoceptor subtypes involved in the NE-induced facilitation of GABAergic transmission, we recorded inhibitory postsynaptic currents (IPSCs) evoked by focal stimulation with paired-pulse (PP) stimuli from PCs in rat cerebellar slices by whole cell recordings and analyzed the PP ratio of the IPSC amplitude. NE increased the IPSC amplitude with a decease in the variance of the PP ratio, which was mimicked by presynaptic manipulation of the transmission caused by increasing the extracellular Ca2+ concentration, confirming that the presynaptic adrenergic receptors are responsible for the facilitation. Pharmacological tests showed that the beta(2)-adrenoceptor antagonist, ICI1 18,551, but not the beta(1)-adrenoceptor antagonist, CGP20712A, blocked the NE-induced IPSC facilitation, suggesting that the beta(2)-adrenoceptors on cerebellar interneurons, basket cells (BCs), mediate the noradrenergic facilitation of GABAergic transmission. Double recordings were performed from BCs and PCs to further characterize the regulation of the GABAergic synapses. First, on-cell recordings from BCs showed that the beta-agonist isoproterenol (ISP) increased the frequencies of the spontaneous spikes in BCs and the spike-triggered IPSCs in PCs recorded with the whole cell mode. The amplitude of the spike-triggered IPSCs decreased or increased depending on the individual GABAergic synapses examined. Forskolin invariably increased both the amplitude and the frequency of the spike-triggered IPSCs. Double whole cell recordings from BC-PC pairs showed that ISP mainly caused an increase in the amplitude of the IPSCs evoked in the PCs by an action current in the BCs produced in response to voltage steps from -60 to -10 mV. Our data suggest that the noradrenergic facilitation of GABAergic transmission in the rat cerebellar cortex is mediated, at least in part, by depolarization and action potential discharges in the BCs through activation of the beta(2)-adrenoceptors in BCs coupled to intracellular cyclic AMP formation.

DOI： 10.1152/jn.2000.84.4.2016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

A single neurotransmitter elicits diverse physiological responses through activation of multiple receptor subtypes and/or heterosynaptic interactions involving distinct synaptic targets. We found that a typical excitatory transmitter released from the climbing fiber (CF) in the cerebellar cortex not only excited Purkinje cells directly but also presynaptically inhibited GABAergic transmission from interneurons converging on the same Purkinje cells. Both homosynaptic and heterosynaptic actions of the CF transmitter (possibly glutamate) were mediated by activation of AMPA receptors. Dual AMPA receptor-mediated functions of excitation and disinhibition may ensure transmission of cerebellar CF signals controlling sensorimotor coordination.

DOI： 10.1038/75718

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The information processing at central synapses is mediated not only by homosynaptic transmission with direct synaptic connections but also by heterosynaptic interactions between distinct synaptic inputs. Using rat brain slices and whole-cell recordings this study aimed to examine the roles of GABA(B) receptors in synaptic interactions in the basolateral amygdala (BLA), a critical brain structure related to fear and anxiety. Stimulation in the BLA produced non-NMDA type glutamate receptor antagonist-sensitive excitatory postsynaptic currents (EPSCs) and bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in the BLA neurons. The GABA(B) receptor agonist baclofen markedly inhibited both EPSCs and IPSCs in a concentration-dependent manner, and the baclofen-induced inhibition was selectively abolished by the GABA(B) receptor antagonist CGP55845A. The paired-pulse ratio of EPSCs and IPSC amplitude was increased by baclofen. The effect of baclofen was mimicked by lowering the external Ca2+ concentration but not by glutamate- and GABA(A)-receptor antagonists. The frequency but not the mean amplitude of miniature EPSCs and IPSCs was decreased by baclofen. The findings suggest that activation of GABA(B) receptors by baclofen reduces the strength of excitatory and inhibitory transmission in the BLA by a presynaptic mechanism. Repetitive conditioning stimulation applied to GABAergic synaptic inputs exerted an inhibitory action on glutamatergic excitatory transmission, and the stimulation-induced inhibition was abolished by CGP55845A. Furthermore, the paired-pulse ratio of EPSCs was increased during the stimulation-induced inhibition. The results in this study provide evidence that synaptic activation of GABA(B) heteroreceptors elicits presynaptic inhibition of glutamatergic excitatory transmission in the BLA. (C) 1999 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0028-3908(99)00126-4

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DOI： 10.1016/S0005-2736(97)00044-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 mu M. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare. (C) 1997 Elsevier Science B.V.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHOTOBIOLOGY  

The photodynamic effects of methylene blue (MB) on wild-type and mutant strains of Paramecium Were studied. From measurements of survival and cell motility under the continuous application of light in the presence of MB, the mutant strains remained alive for about three times longer than the wild-type strain. Although the resting potential of the mutant cells was similar to that of wild-type cells, the continuous photodynamic action shifted the membrane potentials of the mutant and wild-type cells to a depolarized level and a hyperpolarized level, respectively, from that before light application, Under voltage clamping, the mutant cells reduced not only the outward current elicited by the photodynamic action but also the outward tail current elicited by the preceding pulse of hyperpolarization. We conclude that the mutant strain is defective in the activation of Ca2+-dependent K+ channels. This defect might cause a reduction in the Ca2+ influx because of the suppression of the membrane hyperpolarization, which results in the elongation of survival time under the photodynamic action.

DOI： 10.1111/j.1751-1097.1997.tb01941.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHOTOBIOLOGY  

An electrophysiological study of photodynamic action on the Paramecium membrane was carried out. In the presence of methylene blue (MB), light-spot stimulation of an anterior and a posterior part induced a depolarization and a hyperpolarization of the membrane, respectively. Under voltage-clamping, the anterior stimulation induced an inward current, while the posterior stimulation induced an outward current. The amplitudes of these currents were dependent on the membrane potential. When K+ channels were blocked with Cs+ and tetraethyl-ammonium (TEA(+)), the posterior outward current was inhibited. Intracellular application of the Ca2+ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) also inhibited the posterior outward current, but the anterior inward current was unaffected. These results suggest that photodynamic action on the Paramecium membrane primarily opens the Ca2+ channels and the following influx of Ca2+ activates the Ca2+-dependent K+ channels localized mainly on the posterior part of the membrane.

DOI： 10.1111/j.1751-1097.1996.tb09644.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHYSICAL SOC JAPAN  

DOI： 10.1143/JPSJ.62.3351

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：English   Publisher：SPRINGER  

The cooling of deciliated Paramecium cells induced a transient Ca current and its amplitude depended on the rate of the temperature drop. The amplitude of the Ca current was increased by the addition of Ca2+ to the bath solution in a concentration-dependent manner, whereas Ni2+, Co2+, Mn2+ and Mg2+ each reversibly inhibited the Ca current in a concentration-dependent manner with apparent dissociation constants of 0.52, 0.66, 0.67 and 2.17 mmol.l(-1), respectively. The Ca current was also inhibited reversibly by amiloride, with a dissociation constant of 0.32 mmol.l(-1). The Ca current was desensitized by repetitive cooling. The amplitude of the Ca current at the second cooling was smaller than that at the first cooling when the interval was short, but recovered as the interval increased. Replacing extracellular Ca2+ with equimolar Sr2+ or Ba2+ did not significantly affect the amplitude of the current response to cooling, but it accelerated the rate of recovery from desensitization and slowed the decay of the current response. These results suggest that the desensitization and the inactivation of the Ca current may involve a Ca2+-dependent pathway.

DOI： 10.1007/s003590050241

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Language：Japanese  

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