Moritoshi Hirono

GABA(B) receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses.

Metabotropic γ-aminobutyric acid type B (GABAB) and glutamate receptors (mGluRs) are postsynaptically co-expressed at cerebellar parallel fiber (PF)–Purkinje cell (PC) excitatory synapses, but their functional interactions are unclear. We found that mGluR1 agonist-induced currents and [Ca2+]i increases in PCs were enhanced following co-activation of GABAB receptors. A GABAB antagonist and a G-protein uncoupler suppressed these effects. Low-concentration baclofen, a GABAB agonist, augmented mGluR1-mediated excitatory synaptic current produced by stimulating PFs. These results indicate that postsynaptic GABAB receptors functionally interact with mGluR1 and enhance mGluR1-mediated excitatory transmission at PF–PC synapses. The interaction between the two types of metabotropic receptors provides a likely mechanism for regulating cerebellar synaptic plasticity.

Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.

Hair Cells Require Phosphatidylinositol 4,5-Bisphosphate for Mechanical Transduction and Adaptation

After opening in response to mechanical stimuli, hair cell transduction channels adapt with fast and slow mechanisms that each depend on Ca(2+). We demonstrate here that transduction and adaptation require phosphatidylinositol 4,5-bisphosphate (PIP(2)) for normal kinetics. PIP(2) has a striking distribution in hair cells, being excluded from the basal region of hair bundles and apical surfaces of frog saccular hair cells. Localization of a phosphatidylinositol lipid phosphatase, Ptprq, to these PIP(2)-free domains suggests that Ptprq maintains low PIP(2) levels there. Depletion of PIP(2) by inhibition of phosphatidylinositol 4-kinase or sequestration by aminoglycosides reduces the rates of fast and slow adaptation. PIP(2) and other anionic phospholipids bind directly to the IQ domains of myosin-1c, the motor that mediates slow adaptation, permitting a strong interaction with membranes and likely regulating the motors activity. PIP(2) depletion also causes a loss in transduction current. PIP(2) therefore plays an essential role in hair cell adaptation and transduction.

Impaired delay but normal trace eyeblink conditioning in PLCbeta4 mutant mice.

To elucidate the functional role of phospholipase Cβ4 (PLCβ4), which is highly expressed in the Purkinje cells of the rostral cerebellum, cerebellar long-term depression (LTD) and delay and trace eyeblink conditioning were investigated in PLCβ4-deficient mice. Rostral cerebellar LTD and delay eyeblink conditioning were severely impaired, whereas trace eyeblink conditioning was not. These results indicate that PLCβ4 is essential for LTD in the rostral cerebellum and delay conditioning, but not trace conditioning. Rostral cerebellar LTD may be required as a neural substrate for delay conditioning, but is not required for trace conditioning.

Type 1 Inositol Trisphosphate Receptor Regulates Cerebellar Circuits by Maintaining the Spine Morphology of Purkinje Cells in Adult Mice

The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)–PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1flox/flox) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF–PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1flox/flox mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF–PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarboxylase 67

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.

Ethanol enhances both action potential-dependent and action potential-independent GABAergic transmission onto cerebellar Purkinje cells

Ethanol (EtOH) modulates synaptic efficacy in various brain areas, including the cerebellum, which plays a role in motor coordination. Previous studies have shown that EtOH enhances tonic inhibition of cerebellar granule cells, which is one of the possible reasons for the alcohol-induced motor impairment. However, the effects of EtOH on molecular layer interneurons (MLIs) in the mouse cerebellum have remained unknown. Here we found that MLIs were depolarized by EtOH through enhancement of hyperpolarization-activated cationic currents (I(h)). Under physiological conditions, a low EtOH concentration (3-50 mM) caused a small increase in the firing rate of MLIs, whereas, in the presence of blockers for ionotropic glutamate and GABA receptors, EtOH (>or=10 mM) robustly enhanced MLI firing, suggesting that synaptic inputs, which seem to serve as the phasic inhibition, could suppress the EtOH-mediated excitation of MLIs and Purkinje cells (PCs). Even in the absence of synaptic blockers, a high EtOH concentration (100 mM) markedly increased the firing rate of MLIs to enhance GABAergic transmission. Furthermore, 100 mM EtOH-facilitated miniature IPSCs via a mechanism that depended on intracellular cyclic AMP, voltage-dependent Ca(2+) channels, and intracellular Ca(2+) stores, but was independent of I(h) or PKA. The two distinct effects of a high EtOH concentration (>or=100 mM), however, failed to attenuate the EtOH-induced strong depolarization of MLIs. These results suggest that acute exposure to a low EtOH concentration (<or=50 mM) enhanced GABAergic synaptic transmission, which suppressed the EtOH-evoked excitation of MLIs and PCs, thereby maintaining precise synaptic integration of PCs.

IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.

BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells

In myelinated axons, K+ channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na+ channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K+ channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K+ channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni2+ elicited a similar effect on APs, indicating the involvement of Ni2+-sensitive Ca2+ channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex.

Developmental enhancement of α2-adrenoceptor-mediated suppression of inhibitory synaptic transmission onto mouse cerebellar Purkinje cells

Noradrenaline (NA) modulates glutamatergic and GABAergic transmission in various areas of the brain. It is reported that some alpha2-adrenoceptor subtypes are expressed in the cerebellar cortex and alpha2-adrenoceptors may play a role in motor coordination. Our previous study demonstrated that the selective alpha2-adrenoceptor agonist clonidine partially depresses spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse cerebellar Purkinje cells (PCs). Here we found that the inhibitory effect of clonidine on sIPSCs was enhanced during postnatal development. The activation of alpha2-adrenoceptors by clonidine did not affect sIPSCs in PCs at postnatal days (P) 8-10, when PCs showed a few sIPSCs and interneurons in the molecular layer (MLIs) did not cause action potential (AP). In the second postnatal week, the frequency of sIPSCs increased temporarily and reached a plateau at P14. By contrast, MLIs began to fire at P11 with the firing rate gradually increasing thereafter and reaching a plateau at P21. In parallel with this rise in the rate of firing, the magnitude of the clonidine-mediated inhibition of sIPSCs increased during postnatal development. Furthermore, the magnitude of the clonidine-mediated firing suppression in MLIs, which seemed to be mediated by a reduction in amplitude of the hyperpolarization-activated nonselective cation current, I(h), was constant across development. Both alpha2A- and alpha2B-, but not alpha2C-, adrenoceptors were strongly expressed in MLIs at P13, and P31. Therefore, the developmental enhancement of the clonidine-mediated inhibition of sIPSCs is attributed to an age-dependent increase in AP-derived sIPSCs, which can be blocked by clonidine. Thus, presynaptic activation of alpha2-adrenoceptors inhibits cerebellar inhibitory synaptic transmission after the second postnatal week, leading to a restriction of NA signaling, which is mainly mediated by alpha1- and beta2-adrenoceptors in the adult cerebellar neuronal circuit.