Shin’Ichiro Satake

Synaptic activation of AMPA receptors inhibits GABA release from cerebellar interneurons

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.

GABAB receptor-mediated presynaptic inhibition of glutamatergic and GABAergic transmission in the basolateral amygdala

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 EPSC 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.

AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanisms

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 Gi/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 Gi/o‐coupled receptor agonists, baclofen and DCG‐IV, and the P/Q‐type calcium channel blocker ω‐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.

Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3‐deficient heterozygous mice

•  Mutations of ATP1A3 cause rapid‐onset dystonia with parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). •  The mRNA of Atp1a3 was highly expressed in molecular‐layer interneurons and Purkinje cells in the developing mouse cerebellar cortex, and the gene product was observed as dots in the molecular layer, on the surface of Purkinje cell soma and the pinceaux. •  Here we report that Atp1a3+/− mice showed increased symptoms of dystonia when being administrated kainate into cerebellum. We also found enhanced inhibitory neurotransmission between molecular‐layer interneurons and Purkinje cells in the developing cerebellum of Atp1a3+/− mice. •  These findings suggest that ATP1A3 haploinsufficiency in the cerebellum has some effect on the inhibitory, but not the excitatory, circuitry and the interaction among different cell types during development. Disturbances of the cerebellar inhibitory network seem to be the underlying pathophysiological mechanism of dystonia among the increasing spectrum of complex neurological symptoms in RDP and AHC.

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, γ-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.

Paired‐pulse facilitation of multivesicular release and intersynaptic spillover of glutamate at rat cerebellar granule cell–interneurone synapses

•  Paired‐pulse facilitation (PPF) is a widely observed form of presynaptically originated short‐term plasticity. •  Here, we report that, in rat cerebellar cortex, paired‐pulse activation of glutamatergic granule cell axon fibres causes not only a facilitation in the peak amplitude (PPFamp) but also a prolongation in the decay‐time constant (PPPdecay) of the EPSCs recorded from molecular‐layer interneurones (INs). •  PPFamp is elicited, in part, by an increase in the number of released vesicles (that is, multivesicular release), whereas PPPdecay results from extrasynaptic spillover of the transmitter glutamate and its intersynaptic pooling among active synapses, as well as from delayed release as has been explained. •  PPFamp and PPPdecay play unique roles in determining excitability of the INs. •  These findings help us understand the mechanisms underlying encoding and processing of the neuronal information in the cerebellar cortex.

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.

Cav2.1 Channels Control Multivesicular Release by Relying on Their Distance from Exocytotic Ca2+ Sensors at Rat Cerebellar Granule Cells

The concomitant release of multiple numbers of synaptic vesicles [multivesicular release (MVR)] in response to a single presynaptic action potential enhances the flexibility of synaptic transmission. However, the molecular mechanisms underlying MVR at a single CNS synapse remain unclear. Here, we show that the Cav2.1 subtype (P/Q-type) of the voltage-gated calcium channel is specifically responsible for the induction of MVR. In the rat cerebellar cortex, paired-pulse activation of granule cell (GC) ascending fibers leads not only to a facilitation of the peak amplitude (PPFamp) but also to a prolongation of the decay time (PPPdecay) of the EPSCs recorded from molecular layer interneurons. PPFamp is elicited by a transient increase in the number of released vesicles. PPPdecay is highly dependent on MVR and is caused by dual mechanisms: (1) a delayed release and (2) an extrasynaptic spillover of the GC transmitter glutamate and subsequent pooling of the glutamate among active synapses. PPPdecay was specifically suppressed by the Cav2.1 channel blocker ω-agatoxin IVA, while PPFamp responded to Cav2.2/Cav2.3 (N-type/R-type) channel blockers. The membrane-permeable slow Ca2+ chelator EGTA-AM profoundly reduced the decay time constant (τdecay) of the second EPSC; however, it only had a negligible impact on that of the first, thereby eliminating PPPdecay. These results suggest that the distance between presynaptic Cav2.1 channels and exocytotic Ca2+ sensors is a key determinant of MVR. By transducing presynaptic action potential firings into unique Ca2+ signals and vesicle release profiles, Cav2.1 channels contribute to the encoding and processing of neural information.

Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing

The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Cav2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Cav2.1 channels to exocytotic Ca2+ sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex.

Ethanol suppresses climbing fiber-induced presynaptic inhibition of GABAergic transmission at cerebellar interneuron-Purkinje cell synapses

P1-a26 Motilin has a modulatory action on the postsynaptic GABA receptors specifically in the excitatory medial vestibular nuclear neurons Hiroshi Todaka 1 , Tetsuya Tatsukawa 1, Yuchio Yanagawa 3, Katsuei Shibuki 2, Soichi Nagao 1 1 RIKEN, BSI, Lab. for Motor Learning Control, Saitama, Japan 2 Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan 3 Department of Genetic and Behavioral Neuroscience, Gunma University, Gunma, Japan