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

Tabata, Toshihide; Sawada, Satsuki; Araki, Kiyoshi; Bono, Yukiko; Furuya, Shigeki; Kano, Masanobu

doi:10.1016/s0165-0270(00)00323-x

Cited by 77 publications

(82 citation statements)

References 24 publications

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“…Cerebellar neurons were cultured as described previously (Furuya et al, 1998;Tabata et al, 2000). Cerebella from P0 Drp1 flox/flox mice were dissected and incubated with 2 mg/ml papain in HBSS for 30 min at 33°C.…”

Section: Cerebellar Neuron Culturementioning

confidence: 99%

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Kageyama¹,

Zhang²,

Roda³

et al. 2012

J Exp Med

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Section: Cerebellar Neuron Culturementioning

confidence: 99%

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Kageyama¹,

Zhang²,

Roda³

et al. 2012

J Exp Med

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“…for 9-22 days as described (34). In some experiments, pertussis toxin (PTX) or its A-protomer (PTX-AP) was added to the medium Ն13-16 h before recordings.…”

mentioning

confidence: 99%

Ca ²⁺ activity at GABA _B receptors constitutively promotes metabotropic glutamate signaling in the absence of GABA

Tabata

Araishi

Hashimoto

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Type B ␥-aminobutyric acid receptor (GABABR) is a G proteincoupled receptor that regulates neurotransmitter release and neuronal excitability throughout the brain. In various neurons, GABA BRs are concentrated at excitatory synapses. Although these receptors are assumed to respond to GABA spillover from neighboring inhibitory synapses, their function is not fully understood. Here we show a previously undescribed function of GABA BR exerted independent of GABA. In cerebellar Purkinje cells, interaction of GABA BR with extracellular Ca 2؉ (Ca o 2؉ ) leads to a constitutive increase in the glutamate sensitivity of metabotropic glutamate receptor 1 (mGluR1). mGluR1 sensitization is clearly mediated by GABA BR because it is absent in GABABR1 subunitknockout cells. However, the mGluR1 sensitization does not require G i/o proteins that mediate the GABABR's classical functions. Moreover, coimmunoprecipitation reveals complex formation between GABA BR and mGluR1 in the cerebellum. These findings demonstrate that GABA BR can act as Ca o 2؉ -dependent cofactors to enhance neuronal metabotropic glutamate signaling.T he type B ␥-aminobutyric acid receptor (GABA B R) is a G protein-coupled receptor (GPCR) distributed throughout the brain (1-3). GABA B R regulates neurotransmitter release and neuronal excitability via G i/o proteins (4). In the classic view, GABA B R responds to GABA released from inhibitory presynaptic terminals (4). However, in some central neurons including cerebellar Purkinje cells, postsynaptic GABA B Rs are concentrated perisynaptically at the excitatory synapses and present sparsely at the inhibitory synapses (5-7). Because GABA B Rs are insensitive to the excitatory neurotransmitter glutamate, a physiological role of GABA B R at excitatory synapses was assumed to depend on ␥-aminobutyric acid (GABA) spillover from neighboring inhibitory synapses (8, 9).The extracellular domain of GABA B R has an amino acid sequence homology to that of Ca 2ϩ -sensing receptor (CaR) (10). Some studies in the heterologous expression systems (11,12) revealed that GABA B R indeed interacts with extracellular Ca 2ϩ (Ca o 2ϩ ). Point mutation experiments indicate that the proximity of the GABA-binding site of GABA B R1 subunit (GBR1) is responsible for this interaction (11). Although Ca o 2ϩ -GABA B R interaction does not activate G proteins (12), it causes a remarkable conformational change of GABA B R as Ca o 2ϩ allosterically shifts GABA-GABA B R affinity (11,12 (22-25), and developmental synapse elimination (24, 26). In cerebellar Purkinje cells, mGluR1 colocalizes with GABA B R at the annuli of the dendritic spines innervated by excitatory parallel fibers (7,27). We have previously shown that mGluR1 signaling in Purkinje cells is enhanced as a consequence of interaction between Ca o 2ϩ and an unknown surface molecule(s) (28). For the reasons mentioned above, we considered GABA B R as a likely candidate for such a surface molecule.In Purkinje cells, mGluR1 outnumbers the other mGluR subtypes (16) and operates an inward ca...

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“…elongation of a primary dendritic stem, and extensive branching and maturation within the molecular layer (23)(24)(25). Here we identify PKCγ activation and BDNF/TrkB signaling as mediators, respectively, of dendritic growth and synapse formation of postnatally differentiating PNs.…”

Section: Discussionmentioning

confidence: 89%

“…To test this possibility directly, we set up cerebellar slices and dissociated cell cultures in which cells came to resemble postnatal PNs in vivo by developing dendritic branchlets through two successive, tightly regulated stages (23)(24)(25). In the first postnatal week (the normal "early" stage), bipolar fusiform PNs retract their primitive processes and extend numerous short perisomatic protrusions.…”

mentioning

confidence: 99%

Tissue plasminogen activator regulates Purkinje neuron development and survival

Li¹,

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse's 10-fold-increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cγ and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability. plasma membrane integrity | postnatal development | spinocerebellar ataxia | synaptic ultrastructure

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

Cited by 77 publications

References 24 publications

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Ca ²⁺ activity at GABA _B receptors constitutively promotes metabotropic glutamate signaling in the absence of GABA

Tissue plasminogen activator regulates Purkinje neuron development and survival

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

Cited by 77 publications

References 24 publications

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Ca 2+ activity at GABA B receptors constitutively promotes metabotropic glutamate signaling in the absence of GABA

Tissue plasminogen activator regulates Purkinje neuron development and survival

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Ca ²⁺ activity at GABA _B receptors constitutively promotes metabotropic glutamate signaling in the absence of GABA