Topographical distance between presynaptic Ca2+ channels and exocytotic Ca2+ sensors contributes to differential facilitatory actions of roscovitine on neurotransmitter release at cerebellar glutamatergic and GABAergic synapses.

Calcium influx into presynaptic terminals through voltage-gated Ca2+ channels triggers univesicular or multivesicular release of neurotransmitters depending on the characteristics of the release machinery. However, the mechanisms underlying multivesicular release (MVR) and its regulation remain unclear. Previous studies showed that in rat cerebellum, the cyclin-dependent kinase inhibitor roscovitine profoundly increases excitatory postsynaptic current (EPSC) amplitudes at granule cell (GC)-Purkinje cell (PC) synapses by enhancing the MVR of glutamate. This compound can also moderately augment the amplitude and prolong the decay time of inhibitory postsynaptic currents (IPSCs) at molecular layer interneuron (MLI)-PC synapses via MVR enhancement and GABA spillover, thus allowing for persistent activation of perisynaptic GABA receptors. The enhanced MVR may depend on the driving force for Cav 2.1 channel-mediated Ca2+ influx. To determine whether the distinct spatiotemporal dynamics of presynaptic Ca2+ influence MVR, we compared the effects of slow and fast Ca2+ chelators, that is, EGTA and BAPTA, respectively, on roscovitine-induced actions at GC-PC and MLI-PC synapses. Membrane-permeable EGTA-AM decreased GC-PC EPSC and MLI-PC IPSC amplitudes to a similar extent but suppressed the roscovitine-induced enhancement of EPSCs. In contrast, BAPTA-AM attenuated the effects of roscovitine on IPSCs. These results suggest that roscovitine augmented glutamate release by activating the release machinery located distally from the Cav 2.1 channel clusters, while it enhanced GABA release in a manner less dependent on those at distal sites. Therefore, the spatial relationships among Ca2+ channels, buffers, and sensors are critical determinants of the differential facilitatory actions of roscovitine on glutamatergic and GABAergic synapses in the cerebellar cortex.

Roscovitine differentially facilitates cerebellar glutamatergic and GABAergic neurotransmission by enhancing Cav 2.1 channel-mediated multivesicular release.

Synaptic vesicle exocytosis is triggered by Ca2+ influx through several subtypes of voltage-gated calcium channels in the presynaptic terminal. We previously reported that paired-pulse stimulation at brief intervals increases Cav 2.1 (P/Q-type) channel-mediated multivesicular release (MVR) at glutamatergic synapses between granule cells (GCs) and molecular layer interneurons (MLIs) in rat cerebellar slices. However, it has yet to be determined how Cav 2 channel subtypes take part in MVR in single axon terminal. This study therefore aimed at examining the effects of roscovitine on different types of cerebellar synapses that make contacts with Purkinje cells (PCs), because this compound has been shown to enhance Cav 2.1 channel-mediated MVR at GC-MLI synapses. Bath application of roscovitine profoundly increased the amplitude of excitatory postsynaptic currents (EPSCs) at GC-PC synapses by a presynaptic mechanism as previously observed at GC-MLI synapses, whereas it caused a marginal effect on climbing fiber-mediated EPSCs in PCs. At MLI-PC synapses, roscovitine increased both the amplitude and decay time of inhibitory postsynaptic currents (IPSCs) by enhancing multivesicular GABA release. When extracellular Ca2+ concentration ([Ca2+ ]e ) decreased, roscovitine became less effective in increasing GC-PC EPSCs. By contrast, roscovitine was able to augment MLI-PC IPSCs in the low [Ca2+ ]e . The Cav 2.1 channel blocker ω-agatoxin IVA suppressed the roscovitine-induced facilitatory actions on both GC-PC EPSCs and MLI-PC IPSCs. These results demonstrate that roscovitine enhances MVR at the GC-PC excitatory synapses in a manner dependent on the driving force of Cav 2.1 channel-mediated Ca2+ influx into the nerve terminal, while it also facilitates MLI-PC inhibitory transmission via Ca2+ -insensitive mechanisms.

Persistent activation of histamine H1 receptors in the hippocampal CA1 region enhances NMDA receptor-mediated synaptic excitation and long-term potentiation in astrocyte- and D-serine-dependent manner.

Behavioral studies using pharmacological tools have implicated histamine H1 receptors in cognitive function via their interactions with N-methyl-D-aspartate receptors (NMDARs) in the hippocampus. However, little is known about the neurophysiological mechanism that underlies the interaction between H1 receptors and NMDARs. To explore how H1 receptor activation affects hippocampal excitatory neurotransmission and synaptic plasticity, this study aimed to examine the effect of H1 receptor ligands on both NMDAR-mediated synaptic currents and long-term potentiation (LTP) at synapses between Schaffer collaterals and CA1 pyramidal neurons using acute mouse hippocampal slices. We found that the H1 receptor antagonist/inverse agonists, pyrilamine (0.1 µM) and cetirizine (10 µM), decreased the NMDAR-mediated component of stimulation-induced excitatory postsynaptic currents (EPSCs) recorded from CA1 pyramidal neurons without affecting the AMPA receptor-mediated component of EPSCs and its paired pulse ratio. Pretreatment of slices with either the glial metabolism inhibitor, fluoroacetate (5 mM), or D-serine (100 µM) diminished the pyrilamine- or cetirizine-induced attenuation of the NMDAR-mediated EPSCs. Furthermore, the LTP of field excitatory postsynaptic potentials induced following high frequency stimulation of Schaffer collaterals was attenuated with application of pyrilamine or cetirizine. Pretreatment with D-serine again attenuated the pyrilamine-induced suppression of LTP. Our data suggest that H1 receptors in the CA1 can undergo persistent activation induced by their constitutive receptor activity and/or tonic release of endogenous histamine, resulting in facilitation of the NMDAR activity in a manner dependent of astrocytes and the release of D-serine. This led to the enhancement of NMDA-component EPSC and LTP at the Schaffer collateral-CA1 pyramidal neuron synapses.

Pursuit of Neurotransmitter Functions: Being Attracted with Fascination of the Synapse.

In the beginning of the 1970s, only two chemical substances, acetylcholine and γ-aminobutyric acid (GABA), had been definitely established as neurotransmitters. Under such circumstances, I started my scientific career in Professor Masanori Otsuka's lab searching for the transmitter of primary sensory neurons. Until 1976, lines of evidence had accumulated indicating that the undecapeptide substance P could be released as a transmitter from primary afferent fibers into spinal synapses, although the substance P-mediated synaptic response had yet to be identified. Peripheral synapses could serve as a good model and thus, it was demonstrated in the prevertebral sympathetic ganglia by1985 that substance P released from axon collaterals of primary sensory neurons acts as the transmitter mediating non-cholinergic slow excitatory postsynaptic potential (EPSP). At that time, we also found that autonomic synapses were useful to uncover the transmitter role of the opioid peptide enkephalins, whose functions had been unknown since their discovery in 1975. Accordingly, enkephalins were found to serve a transmitter role in mediating presynaptic inhibition of cholinergic fast and non-cholinergic slow transmission in the prevertebral sympathetic ganglia. In 1990s, we attempted to devise a combined technique of brain slices and patch-clamp recordings. We applied it to study the regulatory mechanisms that operate around cerebellar GABAergic inhibitory synapses, because most of the studies then had centered on excitatory synapses and because inhibitory synapses are crucially involved in brain functions and disorders. Consequently, we discovered novel forms of heterosynaptic interactions, dual actions of a single transmitter, and receptor crosstalk, the details of which are described in this review.

Monoaminergic modulation of GABAergic transmission onto cerebellar globular cells.

Cerebellar Purkinje cells (PCs) project their axon collaterals to underneath of the PC layer and make GABAergic synaptic contacts with globular cells, a subgroup of Lugaro cells. GABAergic transmission derived from the PC axon collaterals is so powerful that it could inhibit globular cells and regulate their firing patterns. However, the physiological properties and implications of the GABAergic synapses on globular cells remain unknown. Using whole-cell patch-clamp recordings from globular cells in the mouse cerebellum, we examined the monoaminergic modulation of GABAergic inputs to these cells. Application of either serotonin (5-HT) or noradrenaline (NA) excited globular cells, thereby leading to their firing. The 5-HT- and NA-induced firing was temporally confined and attenuated by GABAergic transmission, although 5-HT and NA exerted an inhibitory effect on the release of GABA from presynaptic terminals of PC axon collaterals. Agonists for 5-HT1B receptors and α2-adrenoceptors mimicked the 5-HT- and NA-induced suppression of GABAergic activity. Through their differential modulatory actions on the cerebellar inhibitory neural circuits, 5-HT facilitated PC firing, whereas NA suppressed it. These results indicate that 5-HT and NA regulate the membrane excitability of globular cells and PCs through their differential modulation of not only the membrane potential but also GABAergic synaptic circuits. Monoaminergic modulation of the neural connections between globular cells and PCs could play a role in cerebellar motor coordination.

Structure-activity relationships of ω-Agatoxin IVA in lipid membranes.

To analyze structural features of ω-Aga IVA, a gating modifier toxin from spider venom, we here investigated the NMR solution structure of ω-Aga IVA within DPC micelles. Under those conditions, the Cys-rich central region of ω-Aga IVA still retains the inhibitor Cys knot motif with three short antiparallel ß-strands seen in water. However, 15N HSQC spectra of ω-Aga IVA within micelles revealed that there are radical changes to the toxin's C-terminal tail and several loops upon binding to micelles. The C-terminal tail of ω-Aga IVA appears to assume a ß-turn like conformation within micelles, though it is disordered in water. Whole-cell patch clamp studies with several ω-Aga IVA analogs indicate that both the hydrophobic C-terminal tail and an Arg patch in the core region of ω-Aga IVA are critical for Cav2.1 blockade. These results suggest that the membrane environment stabilizes the structure of the toxin, enabling it to act in a manner similar to other gating modifier toxins, though its mode of interaction with the membrane and the channel is unique.

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study.

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 GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA 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.

Bakuchiol Is a Phenolic Isoprenoid with Novel Enantiomer-selective Anti-influenza A Virus Activity Involving Nrf2 Activation.

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.

Multiple actions of a D3 dopamine receptor agonist, PD128907, on GABAergic inhibitory transmission between medium spiny neurons in mouse nucleus accumbens shell.

The nucleus accumbens (NAc) plays a crucial role in pathophysiological responses, such as reward-related behaviors, addiction, depression and schizophrenia, through activation of dopaminergic system in the midbrain area. Principal cells in the NAc are medium spiny neurons (MSNs), which constitute the majority (90-95%) of NAc neuron populations in rodents. MSNs are mutually connected to form networks of lateral inhibition. Our previous study showed that activation of D2-like receptors presynaptically inhibited GABAergic transmission between MSN-MSN connections in the NAc. D2-like receptors in MSNs have been reported to consist of D2 and D3 receptors, but their functional roles remain to be elucidated. This study, therefore, aimed at examining the effects of D3 receptor activation on MSN-MSN connections using PD128907, a preferential D3 dopamine receptor agonist, and whole cell recordings from MSNs in acute slices including the NAc. In more than half of cells tested, PD128907 reduced the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner. However, the agonist caused multiple actions, namely, decrease, increase and no significant changes, in the amplitude as well as the frequency of sIPSCs in individual cells. Our data, together with the results from previous studies, show that dopamine could suppress GABAergic transmission, i.e., lateral inhibition between some of MSNs, via activation of both D2 and D3 receptors.

KCC2-mediated regulation of respiration-related rhythmic activity during postnatal development in mouse medulla oblongata.

GABA acts as inhibitory neurotransmitter in the adult central nervous system but as excitatory neurotransmitter during early postnatal development. This shift in GABA's action from excitation to inhibition is caused by a decrease in intracellular chloride concentration ([Cl(-)]i), which in turn is caused by changes in the relative expression levels of the K(+)-Cl(-) co-transporter (KCC2) and the Na(+), K(+)-2Cl(-) co-transporter (NKCC1) proteins. Previous studies have used slices containing the medullary pre-Bötzinger complex (pre-BötC) to record respiration-related rhythmic activity (RRA) from the hypoglossal nucleus (12 N). The role of GABAergic transmission in the regulation of medullary RRA neonatally, however, is yet to be determined. Here, we examined how GABA and chloride co-transporters contribute to RRA during development in the 12 N where inspiratory neurons reside. We recorded extracellular RRA in medullary slices obtained from postnatal day (P) 0-7 mice. RRA was induced by soaking slices in artificial cerebrospinal fluid (aCSF) containing 8mM-K(+). Application of GABA significantly increased the frequency of RRA after P3, whereas application of a KCC2 blocker (R (+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl)oxy]acetic acid (DIOA)) significantly decreased the frequency of RRA after P1. In addition, dense KCC2 immunolabeling was seen in the superior longitudinalis (SL) of the 12 N, which is responsible for retraction of the tongue, from P0 and P7. These results indicate that GABA administration can increase RRA frequency during the first week following birth. This in turn suggests that decreasing [Cl(-)]i levels caused by increasing KCC2 levels in the 12 N could play important roles in regulating the frequency of RRA during development.

Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells.

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.

Activity-dependent coordinated mobility of hippocampal inhibitory synapses visualized with presynaptic and postsynaptic tagged-molecular markers.

Axonal varicosities and dendritic spines at excitatory synapses are dynamic structures essential for synaptic plasticity, whereas the behavior of inhibitory synapses during development and plasticity remains largely unknown. To investigate the morphology and dynamics of inhibitory synapses, we used two distinct pre- and postsynaptic fluorescent probes: one is a yellow fluorescent protein, Venus, incorporated into vesicular GABA transporter (VGAT) gene as a specific marker of presynaptic inhibitory neurons and the other red fluorescent protein (mCherry)-tagged gephyrin, a postsynaptic scaffolding protein, as a postsynaptic marker. Using primary culture of mouse hippocampal neurons and confocal laser-scanning microscopy, we established a system by which close contacts of Venus-positive axonal varicosities with mCherry-labeled gephyrin clusters in the dendritic shafts of dissociated hippocampal pyramidal neurons could be clearly visualized. Time-lapse imaging revealed that: (1) the presynaptic varicosities actively moved with marked changes in their shapes, and the postsynaptic scaffolding protein gephyrin clusters underwent coordinated movements in a tight association with the presynaptic varicosities, (2) the extents of morphological changes and movements depended on the developmental stages, reaching a stable level as the inhibitory synaptic connections matured, and (3) the motility indexes of the varicosity and its counterpart gephyrin cluster were well correlated. Furthermore, action potential blockade with tetrodotoxin treatment reduced the varicosity size, gephyrin cluster mobility as well as the amplitude of GABAergic synaptic currents in pyramidal neurons. Such a neural activity-dependent dynamic change in GABAergic synaptic morphology is likely to play a critical role in the regulatory mechanism underlying the formation and plasticity of inhibitory synapses.

NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms.

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.

The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice.

BACKGROUND: The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. RESULTS: VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. CONCLUSION: VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.

Glutamate transporter EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter mediating inhibition of GABA release from interneurons.

Neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to exert concerted control of the synaptic strength within neural pathways that converge on single target neurons. The excitatory transmitter released from climbing fibers (CFs), presumably glutamate, is shown to inhibit γ-aminobutyric acid (GABA) release at basket cell (BC)-Purkinje cell (PC) synapses in the rat cerebellar cortex through its extrasynaptic diffusion and activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on BC axon terminals. This study aimed at examining how the CF transmitter-diffusion-mediated presynaptic inhibition is controlled by glutamate transporters. Pharmacological blockade of the PC-selective neuronal transporter EAAT4 markedly enhanced CF-induced inhibition of GABAergic transmission. Tetanic CF-stimulation elicited long-term potentiation of glutamate transporters in PCs, and thereby attenuated the CF-induced inhibition. Combined use of electrophysiology and immunohistochemistry revealed a significant inverse relationship between the level of EAAT4 expression and the inhibitory action of CF-stimulation on the GABA release at different cerebellar lobules - the CF-induced inhibition was profound in lobule III, where the EAAT4 expression level was low, whereas it was minimal in lobule X, where EAAT4 was abundant. The findings clearly demonstrate that the neuronal glutamate transporter EAAT4 in PCs plays a critical role in the extrasynaptic diffusion of CF transmitter - it appears not only to retrogradely determine the degree of CF-mediated inhibition of GABAergic inputs to the PC by controlling the glutamate concentration for intersynaptic diffusion, but also regulate synaptic information processing in the cerebellar cortex depending on its differential regional distribution as well as use-dependent plasticity of uptake efficacy.

Characterization of AMPA receptors targeted by the climbing fiber transmitter mediating presynaptic inhibition of GABAergic transmission at cerebellar interneuron-Purkinje cell synapses.

The climbing fiber (CF) neurotransmitter not only excites the postsynaptic Purkinje cell (PC) but also suppresses GABA release from inhibitory interneurons converging onto the same PC depending on AMPA-type glutamate receptor (AMPAR) activation. Although the CF-/AMPAR-mediated inhibition of GABA release provides a likely mechanism boosting the CF input-derived excitation, how the CF transmitter reaches target AMPARs to elicit this action remains unknown. Here, we report that the CF transmitter diffused from its release sites directly targets GluR2/GluR3 AMPARs on interneuron terminals to inhibit GABA release. A weak GluR3-AMPAR agonist, bromohomoibotenic acid, produced excitatory currents in the postsynaptic PCs without presynaptic inhibitory effect on GABAergic transmission. Conversely, a specific inhibitor of the GluR2-lacking/Ca2+-permeable AMPARs, philanthotoxin-433, did not affect the CF-induced inhibition but suppressed AMPAR-mediated currents in Bergmann glia. A low-affinity GluR antagonist, gamma-D-glutamylglycine, or retardation of neurotransmitter diffusion by dextran reduced the inhibitory action of CF-stimulation, whereas blockade of glutamate transporters enhanced the CF-induced inhibition. The results suggest that the CF transmitter released after repeated stimulation overwhelms local glutamate uptake and thereby diffuses from the release site to reach GluR2/GluR3 AMPARs on nearby interneuron terminals. Double immunostaining showed that GluR2/3 subunits and glutamate decarboxylase or synaptophysin are colocalized at the perisomatic GABAergic processes surrounding PCs. Finally, electron microscopy detected specific immunoreactivity for GluR2/3 at the presynaptic terminals of symmetric axosomatic synapses on the PC. These findings demonstrate that the CF transmitter directly inhibits GABA release from interneurons to the PC, relying on extrasynaptic diffusion and local heterogeneity in AMPAR subunit compositions.

Distribution and pharmacological characterization of primate NK-1 and NK-3 tachykinin receptors in the central nervous system of the rhesus monkey.

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. 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. 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. 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. 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.

Modulation of presynaptic Ca2+ entry by AMPA receptors at individual GABAergic synapses in the cerebellum.

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 Ca2+ channels, eliciting a brief 3-5 microM transient, followed by a longer-term, 15-30 nM rise of free Ca2+ (above the resting level of approximately 100 nM). Brief activation of local AMPA receptors suppresses Ca2+ entry (probably by silencing 20-40 P/Q-type channels) in a subgroup of terminals that tend to show a higher dynamic range of Ca2+ signaling. The results provide the first quantitative description of presynaptic Ca2+ kinetics and its modulation by AMPA receptor activation (most likely via a glutamate spillover-mediated mechanism) at identified GABAergic synapses.

beta-Adrenoceptor-mediated long-term up-regulation of the release machinery at rat cerebellar GABAergic synapses.

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 LTP 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.

Metabotropic P2Y purinoceptor-mediated presynaptic and postsynaptic enhancement of cerebellar GABAergic transmission.

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 Gq-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 GABAA receptor-mediated IPSCs in PCs in a manner sensitive to a P2Y1R antagonist, N6-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.