Shin-ya Kawaguchi

Opposing roles in neurite growth control by two seven-pass transmembrane cadherins

The growth of neurites (axon and dendrite) should be appropriately regulated by their interactions in the development of nervous systems where a myriad of neurons and their neurites are tightly packed. We show here that mammalian seven-pass transmembrane cadherins Celsr2 and Celsr3 are activated by their homophilic interactions and regulate neurite growth in an opposing manner. Both gene-silencing and coculture assay with rat neuron cultures showed that Celsr2 enhanced neurite growth, whereas Celsr3 suppressed it, and that their opposite functions were most likely the result of a difference of a single amino acid residue in the transmembrane domain. Together with calcium imaging and pharmacological analyses, our results suggest that Celsr2 and Celsr3 fulfill their functions through second messengers, and that differences in the activities of the homologs results in opposite effects in neurite growth regulation.

Sustained Structural Change of GABAA Receptor-Associated Protein Underlies Long-Term Potentiation at Inhibitory Synapses on a Cerebellar Purkinje Neuron

Fast inhibitory synaptic transmission is predominantly mediated by GABAA receptor (GABAAR) in the CNS. Although several types of neuronal activity-dependent plasticity at GABAergic synapses have been reported, the detailed mechanism is elusive. Here we show that binding of structurally altered GABAAR-associated protein (GABARAP) to GABAAR γ2 subunit and to tubulin is critical for long-term potentiation [called rebound potentiation (RP)] at inhibitory synapses on a cerebellar Purkinje neuron (PN). Either inhibition of GABARAP association with GABAARγ2 or deletion of tubulin binding region of GABARAP impaired RP. Inhibition of tubulin polymerization also suppressed RP. Thus, precise regulation of GABAARγ2–GABARAP–microtubule interaction is critical for RP. Furthermore, competitive inhibition of GABARAP binding to GABAARγ2 after the RP establishment attenuated the potentiated response, suggesting that GABARAP is critical not only for the induction but also for the maintenance of RP. Fluorescence resonance energy transfer analysis revealed that GABARAP underwent sustained structural alteration after brief depolarization of a PN depending on the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is required for the RP induction. The susceptibility of GABARAP to undergo structural alteration was abolished by an amino acid replacement in GABARAP. Furthermore, RP was impaired by expression of the mutant GABARAP with the replacement. Together, we conclude that GABAAR association with structurally altered GABARAP downstream of CaMKII activation is essential for RP.

Suppression of inhibitory synaptic potentiation by presynaptic activity through postsynaptic GABA(B) receptors in a Purkinje neuron.

At inhibitory synapses on a cerebellar Purkinje neuron, the depolarization caused by heterosynaptic climbing fiber activation induces long-lasting potentiation accompanied by an increase in GABA(A) receptor responsiveness. Here we show that activation of a presynaptic inhibitory interneuron during the conditioning postsynaptic depolarization suppresses the potentiation. The suppression is due to postsynaptic GABA(B) receptor activation by GABA released from presynaptic terminals. The results suggest that GABA(B) receptor activation decreases the activity of cAMP-dependent protein kinase through the G(i)/G(o) proteins. The presynaptic activity-dependent suppression of synaptic plasticity is a novel regulatory mechanism of synaptic efficacy at individual synapses and may contribute to the learning and computational ability of the cerebellar cortex.

Impaired cerebellar functions in mutant mice lacking DNER.

DNER is a transmembrane protein carrying extracellular EGF repeats and is strongly expressed in Purkinje cells (PCs) in the cerebellum. Current study indicated that DNER functions as a new Notch ligand and mediates the functional communication via cell-cell interaction. By producing and analyzing knockout mice lacking DNER, we demonstrate its essential roles in functional and morphological maturation of the cerebellum. The knockout mice exhibited motor discoordination in the fixed bar and rota-rod tests. The cerebellum from the knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization. Histochemical and electrophysiological analyses detected that PCs retained multiple innervations from climbing fibers (CFs) in the mutant cerebellum. Synaptic transmission from parallel fibers (PFs) or CFs to PCs was apparently normal, while glutamate clearance at the PF-PC synapses was significantly impaired in the mutant mice. Moreover, the protein level of GLAST, the glutamate transporter predominantly expressed in Bergmann glia (BG), was reduced in the mutant cerebellum. Our results indicate that DNER takes part in stimulation of BG maturation via intercellular communication and is essential for precise cerebellar development.

Long-term Potentiation of Inhibitory Synaptic Transmission onto Cerebellar Purkinje Neurons Contributes to Adaptation of Vestibulo-Ocular Reflex

Synaptic plasticity in the cerebellum is thought to contribute to motor learning. In particular, long-term depression (LTD) at parallel fiber (PF) to Purkinje neuron (PN) excitatory synapses has attracted much attention of neuroscientists as a primary cellular mechanism for motor learning. In contrast, roles of plasticity at cerebellar inhibitory synapses in vivo remain unknown. Here, we have investigated the roles of long-lasting enhancement of transmission at GABAergic synapses on a PN that is known as rebound potentiation (RP). Previous studies demonstrated that binding of GABAA receptor with GABAA receptor-associated protein (GABARAP) is required for RP, and that a peptide that blocks this binding suppresses RP induction. To address the functional roles of RP, we generated transgenic mice that express this peptide fused to a fluorescent protein selectively in PNs using the PN-specific L7 promoter. These mice failed to show RP, although they showed no changes in the basal amplitude or frequency of miniature IPSCs. The transgenic mice also showed no abnormality in gross cerebellar morphology, LTD, or other excitatory synaptic properties, or intrinsic excitability of PNs. Next, we attempted to evaluate their motor control and learning ability by examining reflex eye movements. The basal dynamic properties of the vestibulo-ocular reflex and optokinetic response, and adaptation of the latter, were normal in the transgenic mice. In contrast, the transgenic mice showed defects in the adaptation of vestibulo-ocular reflex, a model paradigm of cerebellum-dependent motor learning. These results together suggest that RP contributes to a certain type of motor learning.

Control of Inhibitory Synaptic Outputs by Low Excitability of Axon Terminals Revealed by Direct Recording

An axon is thought to faithfully conduct action potentials to its terminals. However, many features of the axon and axon terminals, especially at inhibitory synapses, remain unknown. By directly recording from the axon and terminal of a cultured cerebellar Purkinje cell (PC), we demonstrate that low membrane excitability of axon terminals shapes synaptic output. Simultaneous measurements of presynaptic capacitance and evoked IPSCs revealed PC axon terminals contained large readily releasable synaptic vesicles that exhibited a low release probability. Nevertheless, IPSCs evoked by stimulating a PC soma underwent frequency-dependent depression. Direct axonal recordings showed that high-frequency action potentials were faithfully conducted over axonal bifurcations but were attenuated around terminals. Sparse Na(+) channels relative to enriched voltage-gated K(+) channels in terminals caused short-term depression of IPSCs by reducing Ca(2+) influx. Together with confirmation in slice recordings, our findings reveal a presynaptic mechanism that shapes short-term synaptic depression without depleting releasable vesicles.

Gating of long-term depression by Ca2+/calmodulin-dependent protein kinase II through enhanced cGMP signalling in cerebellar Purkinje cells.

•  Long‐term depression (LTD) at parallel fibre synapses on a cerebellar Purkinje cell has been regarded as a cellular basis for motor learning. •  Although Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has been implicated in the LTD induction and motor learning, the underlying molecular mechanism remains unclear. •  By combined application of simulation and experiments, we have attempted to explore the potential signalling pathway underlying the CaMKII involvement in LTD. Our data show that CaMKII supports the LTD‐inducing signalling pathway consisting of other protein kinases such as protein kinase C and mitogen‐activated protein kinase. •  The gating of the LTD‐inducing pathway by CaMKII is mediated by negative regulation of phosphodiesterase 1, and the resultant facilitation of the cGMP/protein kinase G (PKG) pathway. As a result, protein phosphatase 2A activity was suppressed, supporting the LTD induction. •  In addition, nitric oxide‐mediated cGMP/PKG activation compensated for the lack of CaMKII activation in LTD induction. •  This study provides a comprehensive understanding of elaborate intracellular signalling mechanisms for LTD regulation.

Roles of Inhibitory Interneurons in the Cerebellar Cortex

Abstract: The roles of inhibitory interneurons in the cerebellar cortex were investigated. First, Golgi cells were specifically eliminated in transgenic mice in which Golgi cells expressed human interleukin‐2 receptor α subunit (IL2Rα). Injection of exotoxin coupled to anti‐IL2Rα antibody in the cerebellum of the transgenic mouse eliminated Golgi cells and abolished GABA and synaptic inhibition in the granular layer. After elimination of Golgi cells, acute severe ataxia and subsequent mild motor discoordination were observed. In the latter chronic phase, NMDA receptor‐mediated synaptic response was reduced in granule cells. Our findings indicate that elimination of GABAergic inhibition in the granular layer caused overexcitation of granule cells resulting in severe ataxia, and then NMDA receptors in granule cells were downregulated, compensating for the reduction of GABAergic inhibition and improving motor control. In the second part, we report on the regulation mechanism of synaptic plasticity at inhibitory synapses on Purkinje cells (PCs). Inhibitory synaptic transmission on a PC is potentiated after repetitive PC depolarization. This synaptic plasticity (rebound potentiation, RP) was suppressed when a presynaptic neuron was activated during the PC depolarization. This synaptic regulation is unique in the sense that the homosynaptic activity suppresses the induction of synaptic plasticity. The mechanism of how presynaptic activity suppresses RP was examined. GABA released from the presynaptic terminal activated not only GABAA receptor but also GABAB receptor. The latter was coupled to Gi/o proteins, which downregulated adenylyl cyclase reducing cAMP and inactivated cAMP‐dependent protein kinase (PKA). Downregulation of PKA suppressed RP induction.

mGluR1-mediated facilitation of long-term potentiation at inhibitory synapses on a cerebellar Purkinje neuron

Synaptic plasticity has been studied extensively at excitatory synapses, whereas studies on plasticity at GABAergic inhibitory synapses have been limited. In the rat cerebellar cortex, postsynaptic depolarization of a Purkinje neuron (PN) induces long‐term potentiation of GABAA receptor (GABAAR) responsiveness (termed rebound potentiation; RP). Induction of RP requires an increase in intracellular Ca2+ concentration and resultant activation of Ca2+/calmodulin‐dependent protein kinase II (CaMKII). We previously reported that GABAB receptor (GABABR) activation coupled with depolarization suppresses RP induction by facilitating protein phosphatase 1 (PP‐1)‐mediated inhibition of CaMKII through down‐regulation of cAMP‐dependent protein kinase A (PKA) activity. Here, we examined the involvement of metabotropic glutamate receptor type 1 (mGluR1) in RP regulation. RP was monitored with the amplitudes of either the current responses to GABA or miniature inhibitory postsynaptic currents recorded from a PN in a primary culture or in a cerebellar slice. Inhibition of mGluR1 by an antagonist, 7(hydroxyimino)cyclopropa[b]chromen‐1a‐carboxylate‐ethyl‐ester (CPCCOEt), prevented RP induction, which was abolished either by activation of adenylyl cyclase or by inhibition of PP‐1. Furthermore, mGluR1 inhibition impaired depolarization‐induced CaMKII activation. By contrast, activation of mGluR1 by the agonist (R,S)3,5‐dihydroxyphenylglycine (DHPG) rescued RP induction from its suppression by GABABR activation. The rescue was impaired either by inhibition of PKA or by facilitation of PP‐1 activity. In addition, mGluR1 activation counteracted the GABABR‐mediated CaMKII inhibition. Taken together, these results suggest that mGluR1 activity counteracts GABABR activity and contributes to RP induction through PKA activation, down‐regulation of PP‐1 and up‐regulation of CaMKII.

Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Synaptic plasticity, neuronal activity‐dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive‐feedback signaling pathways by an increase in intracellular Ca2+ concentration ([Ca2+]i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca2+]i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca2+/calmodulin‐dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive‐feedback reactions, CaMKII autophosphorylation and CaMKII‐mediated inhibition of a CaM‐dependent phosphodiesterase, PDE1. The simulation predicted that PDE1‐mediated feedforward inhibition of CaMKII predominantly controls the Ca2+ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca2+ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1.