Migration of Interneuron Precursors in the Nascent Cerebellar Cortex.

The cerebellum arguably constitutes one of the best characterized central nervous circuits, and its structure, cellular function, and histogenesis have been described in exceptional quantitative detail. A notable exception to this is the development of its inhibitory interneurons, and in particular the extensive migrations of future basket and stellate cells. Here, we used acute slices from 8-day-old mice to assess the migration of Pax2-EGFP-tagged precursors of these cells en route to the molecular layer during their transit through the nascent cerebellar cortex. We document that movement of these cells is highly directed. Their speed and directional persistence are larger in the nascent granule cell layer than in the molecular layer. And they migrate periodically, with periods of effective, directed translocation separated by bouts of rather local movement. Finally, we document that the arrangement of these cells in the adult molecular layer is characterized by clustering. These data are discussed with a focus on potential generative mechanisms for the developmental pattern observed.

Synaptic input as a directional cue for migrating interneuron precursors.

During CNS development, interneuron precursors have to migrate extensively before they integrate in specific microcircuits. Known regulators of neuronal motility include classical neurotransmitters, yet the mechanisms that assure interneuron dispersal and interneuron/projection neuron matching during histogenesis remain largely elusive. We combined time-lapse video microscopy and electrophysiological analysis of the nascent cerebellum of transgenic Pax2-EGFP mice to address this issue. We found that cerebellar interneuronal precursors regularly show spontaneous postsynaptic currents, indicative of synaptic innervation, well before settling in the molecular layer. In keeping with the sensitivity of these cells to neurotransmitters, ablation of synaptic communication by blocking vesicular release in acute slices of developing cerebella slows migration. Significantly, abrogation of exocytosis primarily impedes the directional persistence of migratory interneuronal precursors. These results establish an unprecedented function of the early synaptic innervation of migrating neuronal precursors and demonstrate a role for synapses in the regulation of migration and pathfinding.

Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.

Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hallmarks of expansion of stem and somatic cells, and enables distinction from cell cycle variations. High-resolution microscopy in embryonic heart and brain tissues of enhanced green fluorescent protein-anillin transgenic mice allows live monitoring of cell division and quantitation of cell cycle kinetics. Analysis of cell division in hearts post injury shows that border zone cardiomyocytes in the infarct respond with increasing ploidy, but not cell division. Thus, the enhanced green fluorescent protein-anillin system enables monitoring and measurement of cell division in vivo and markedly simplifies in vitro analysis in fixed cells.

Gray matter NG2 cells display multiple Ca2+-signaling pathways and highly motile processes.

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca(2+)-concentration ([Ca(2+)](i)) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca(2+)-signaling pathways we combined patch-clamp recordings, Ca(2+)-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca(2+)-channels, Ca(2+)-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca(2+)](i)-elevations in NG2 cells. The Ca(2+)-influx is amplified by Ca(2+)-induced Ca(2+)-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca(2+)](i) elevations, suggesting that [Ca(2+)](i) elevations in NG2 cells might be restricted to their processes. Local Ca(2+)-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.

Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR.

The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.