Toshihide Tabata

A reliable method for culture of dissociated mouse cerebellar cells enriched for Purkinje neurons

The cerebellar Purkinje neuron (PN) serves as an important model in studies of neuronal development in the mammalian central nervous system. Dissociated PN preparations maintained in an in-vitro environment with simplified cellular and biochemical conditions can facilitate molecular analyses of neuronal development. Here we describe a reliable method to prepare dissociated cultures of mouse cerebellar neurons maintained with a serum-free, Dulbeccos modified Eagles medium/F-12 nutrient-based medium, which facilitates PN survival and dendritic differentiation. The survival of mouse PNs in this culture was maximized when cerebellar cells were (1) taken from prenatal animals, (2) dissociated with papain, and (3) seeded at a density of 5 000 000 cells/ml or higher. Dissociated PNs prepared by our method from mice of embryonic day 18 (E 18) reproduced several morphological and electrophysiological changes seen in intact postnatal rodents with similar time-courses. Therefore, our culture method offers a useful model for investigating molecular mechanisms underlying postnatal neuronal development.

Postsynaptic GABAB receptor signalling enhances LTD in mouse cerebellar Purkinje cells.

Long‐term depression (LTD) of excitatory transmission at cerebellar parallel fibre–Purkinje cell synapses is a form of synaptic plasticity crucial for cerebellar motor learning. Around the postsynaptic membrane of these synapses, B‐type γ‐aminobutyric acid receptor (GABABR), a Gi/o protein‐coupled receptor for the inhibitory transmitter GABA is concentrated and closely associated with type‐1 metabotropic glutamate receptors (mGluR1) whose signalling is a key factor for inducing LTD. We found that in cultured Purkinje cells, GABABR activation enhanced LTD of a glutamate‐evoked current (LTDglu), increasing the magnitude of depression. It has been reported that parallel fibre–Purkinje cell synapses receive a micromolar level of GABA spilt over from the synaptic terminals of the neighbouring GABAergic interneurons. This level of GABA was able to enhance LTDglu. Our pharmacological analyses revealed that the βγ subunits but not the α subunit of Gi/o protein mediated GABABR‐mediated LTDglu enhancement. Gi/o protein activation was sufficient to enhance LTDglu. In this respect, LTDglu enhancement is clearly distinguished from the previously reported GABABR‐mediated augmentation of an mGluR1‐coupled slow excitatory postsynaptic potential. Baclofen application for only the induction period of LTDglu was sufficient to enhance LTDglu, suggesting that GABABR signalling may modulate mechanisms underlying LTDglu induction. Baclofen augmented mGluR1‐coupled Ca2+ release from the intracellular stores in a Gi/o protein‐dependent manner. Therefore, GABABR‐mediated LTDglu enhancement is likely to result from augmentation of mGluR1 signalling. Furthermore, pharmacological inhibition of GABABR reduced the magnitude of LTD at parallel fibre–Purkinje cell synapses in cerebellar slices. These findings demonstrate a novel mechanism that would facilitate cerebellar motor learning.

Extracellular calcium controls the dynamic range of neuronal metabotropic glutamate receptor responses.

The metabotropicglutamate receptors (mGluRs) are neurotransmitter receptors important for synaptic plasticity in the brain. Here we report that native mGluR-mediated neuronal responses to glutamate are profoundly modulated by extracellular calcium (Ca2+(o)). In mouse cerebellar Purkinje cells (PCs), Ca2+(o) drastically broadened the effective dose range for glutamate analogs in which native mGluR1-mediated cation current and intracellular Ca2+ mobilization were evoked. This effect has not been observed for recombinant mGluRs expressed in the heterologous cell systems. Ca2+(o) also drastically augmented these native mGluR-mediated responses to the glutamate analog. These Ca2+(o) effects were observed in both the wild-type mice and the mutant mice expressing mGluR1 specifically in their PCs, suggesting that the native mGluR1 in the PCs but not those in other cell types are the key mediators of the effects. These findings demonstrate that Ca2+(o) plays an important role in regulating native mGluR-mediated neuronal responses.

Insulin-like growth factor-I as a promoting factor for cerebellar Purkinje cell development

In the mammalian CNS, the peptide hormone insulin‐like growth factor‐I (IGF‐I) is synthesized in a certain subset of neurons and, it has been suggested, serves as a local neurotrophic factor. A postnatal increase in the expression of IGF‐I and the type‐1 IGF receptors (IGFR1) in the cerebellar cortex and its related brain regions indicates that developing cerebellar Purkinje cells (PC) may be an important target of IGF‐I. However, little is known about how IGF‐I influences PC development. Here we addressed this question, using a reduced environment of cerebellar neuron culture derived from perinatal mice. IGF‐I exogenously applied at a physiological concentration (10 nm) greatly promoted the dendritic growth and survival of the PCs. By contrast, IGF‐I only slightly promoted the somatic growth and little affected the maturation of the electrophysiological excitability of the PCs. The closely related hormone insulin had weaker promoting effects than did IGF‐I. IGF‐I appeared to at least bind to IGFR1 and to up‐regulate the signalling pathways involving the phosphoinositide 3‐kinase (PI3‐K), mitogen‐activated protein kinase (MAPK), p38 kinase (p38K), and an unknown signalling molecule(s). These signalling pathways may be coupled to the individual aspects of PC development in different manners and this may explain the difference in effects of IGF‐I among these aspects. These findings suggest that IGF‐I serves as a promoting factor for PC development, particularly postnatal survival and dendritic growth.

G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cells.

Adenosine receptors (ARs) are G protein‐coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between Gi/o protein‐coupled A1‐subtype AR (A1R) and type‐1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated‐patch voltage‐clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1‐coupled inward current. A1R agonists also depressed mGluR1‐coupled intracellular Ca2+ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist‐induced depression persisted after blockade of Gi/o protein. The depression appeared to involve neither the cAMP‐protein kinase A cascade downstream of the alpha subunits of Gi/o and Gs proteins, nor cytoplasmic Ca2+ that is suggested to be regulated by the beta‐gamma subunit complex of Gi/o protein. Moreover, A1R did not appear to affect Gq protein which mediates the mGluR1‐coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R‐mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R‐mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons.

Calcium dependence of native metabotropic glutamate receptor signaling in central neurons

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that are distributed throughout the brain and play important roles in regulation of synaptic efficacy. Some studies report that mGluRs heterologously expressed in nonneuronal cells are sensitive not only to glutamate but also to extracellular Ca2+ (Cao2+). We studied the Cao2+-sensitivity of native mGluRs in mammalian central neurons. In cerebellar Purkinje cells that naturally express type-1 mGluR (mGluR1), physiological levels of Cao2+ (around 2 mM) activate mGluR1-mediated intracellular Ca2+ mobilization. The activation of the native mGluR1 response to Cao2+ appears to be slower than that to glutamate. Cao2+ (2 mM) also augments glutamate analog-evoked, native mGluR1-mediated inward cation current and intracellular Cao2+ mobilization. Detailed analysis of this effect suggests that Cao2+ modulates the glutamate responsiveness of native and heterologously expressed mGluR1s in different manners. These findings suggest that Cao2+ may enhance the basal level and glutamate responsiveness of neuronal mGluR signaling in vivo.

GABAergic activation of an inwardly rectifying K+ current in mouse cerebellar Purkinje cells

Cerebellar Purkinje cells integrate motor information conveyed by excitatory synaptic inputs from parallel and climbing fibres. Purkinje cells abundantly express B‐type G‐protein‐coupled γ‐aminobutyric acid receptors (GABABR) that are assumed to mediate major responses, including postsynaptic modulation of the synaptic inputs. However, the identity and function of effectors operated by GABABR are not fully elucidated. Here we characterized an inwardly rectifying current activated by baclofen (Ibacl), a GABABR agonist, in cultured mouse Purkinje cells using a ruptured‐patch whole‐cell technique. Ibacl is operated by GABABR via Gi/o‐proteins, as it is not inducible in pertussis‐toxin‐pretreated cells. Ibacl is carried by K+ because its reversal potential shifts with the equilibrium potential of K+. Ibacl is blocked by 10−3m Ba2+ or Cs+, and 10−8m tertiapin‐Q. Upon the onset and offset of a hyperpolarizing step, Ibacl is activated and deactivated, respectively, with double‐exponential time courses (time constants, <1 ms and 30–80 ms). Based on similarities in the above properties, G‐protein‐coupled inwardly rectifying K+ (GIRK) channels are thought to be responsible for Ibacl. Perforated‐patch recordings from cultured Purkinje cells demonstrate that Ibacl hyperpolarizes the resting potential and the peak level achieved by glutamate‐evoked potentials initiated in the dendrites. Moreover, cell‐attached recordings from Purkinje cells in cerebellar slices demonstrate that Ibacl impedes spontaneous firing. Therefore, Ibacl may reduce the postsynaptic and intrinsic excitability of Purkinje cells under physiological conditions. These findings give a new insight into the role of GABABR signalling in cerebellar information processing.

Altered agonist sensitivity and desensitization of neuronal mGluR1 responses in knock‐in mice by a single amino acid substitution at the PKC phosphorylation site

mGluR1 and mGluR5 of the metabotropic glutamate receptor family are coupled to inositol trisphosphate–Ca2+ signal cascades and evoke distinct Ca2+ responses in neural cells and heterologously expressing cells. In heterologous cells, stimulation of recombinant mGluR1 evokes a single‐peaked Ca2+ response whereas mGluR5 elicits an oscillatory Ca2+ response. The distinct Ca2+ responses are interchangeable by single amino substitution of aspartate for threonine at the corresponding position of the carboxy‐terminal cytoplasmic regions of mGluR1 and mGluR5, respectively. In this investigation, we generated knock‐in mice, termed mGluR1 D854T mice, in which aspartate of mGluR1 was replaced with threonine. We examined the effect of this D854T substitution on Ca2+ and current responses mediated by mGluR1 in cultured cerebellar Purkinje cells. Stimulation of mGluR1 D854T by a group 1 mGluR agonist, 3,5‐dihydroxyphenylglycine (DHPG) evoked, as in wild‐type mGluR1, only single‐peaked Ca2+ responses as measured by Ca2+ fluorometric analysis. We then examined mGluR1‐induced inward currents carried by nonselective cation channels during whole‐cell recordings from cultured Purkinje cells. The mGluR1 D854T mutation abolished the responsiveness of mGluR1 to low concentrations of DHPG (0.5–500 nm) and reduced its desensitization during prolonged agonist application. mGluR1 D854T homozygous mutants showed no apparent behavioural abnormality as analysed by motor movement tests. These results indicate that, although additional modulatory mechanisms seem to be required to produce oscillatory Ca2+ responses of mGluR1, the single amino acid substitution at position 854 of mGluR1 is capable of influencing the kinetics of neuronal mGluR1 responses, most probably through PKC‐mediated phosphorylation.

GABA(B) receptor-mediated modulation of glutamate signaling in cerebellar Purkinje cells.

Since Purkinje cells are the sole output neurons of the cerebellar cortex, the postsynaptic integration of excitatory and inhibitory synaptic inputs in this cell type is a pivotal step for cerebellar motor information processing. In Purkinje cells, Gi/o protein-coupled B-type γ-aminobutyric acid receptor (GABABR) is expressed at the annuli of the dendritic spines that are innervated by the glutamatergic terminals of parallel fibers. The subcellular localization of GABABR suggests the possibility of postsynaptic interplay between GABABR and glutamate signaling. It has recently been demonstrated that GABABR indeed modulates α amino-3-hydroxy-5-methyl-4-isoxalone propionate-type ionotropic glutamate receptor (AMPAR)-mediated and type-1 metabotropic glutamate receptor (mGluR1)-mediated signaling. Interestingly, GABABR exerts modulatory actions not only via the classical Gi/o protein-dependent signaling cascade but also via a Gi/o protein-independent interaction between GABABR and mGluR1. In this review, we compare the physiological nature, underlying mechanisms, and possible functional significance of these modulatory actions of GABABR