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We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted...
The trp (transient receptor potential) gene encodes a Ca2+ channel responsible for the major component of the phospholipase C (PLC) mediated light response in Drosophila. In trp mutants, maintained light leads to response decay and temporary total loss of sensitivity (inactivation). Using genetically targeted PIP2-sensitive inward rectifier channels (Kir2.1) as biosensors, we provide evidence that...
Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally...
Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component...
Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibited exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibited exocytosis less effectively after priming. We propose that SNAREs...
Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase (SOD1G37R) linked to amyotrophic lateral sclerosis...
Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of ∼40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the...
Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic...
On a radial temperature gradient, C. elegans worms migrate, after conditioning with food, toward their cultivation temperature and move along this isotherm. This experience-dependent behavior is called isothermal tracking (IT). Here we show that the neuron-specific calcium sensor-1 (NCS-1) is essential for optimal IT. ncs-1 knockout animals show major defects in IT behavior, although their chemotactic,...
Newly synthesized Arc mRNA is selectively targeted to synapses that have experienced particular patterns of activity. Here, we demonstrate that the targeting requires NMDA receptor activation. Arc expression was induced by an electroconvulsive seizure, and the newly synthesized mRNA was then targeted to synaptic sites by activating the perforant path projections to the dentate gyrus. When micropipette...
Cortical interneurons arise from the proliferative zone of the ventral telencephalon, the ganglionic eminence, and migrate into the developing neocortex. The spatial patterns of migratory interneurons reflect the complementary expression of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, MET, in the forebrain. Scatter assays on forebrain explants demonstrate regionally specific...
Nav1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Nav1.2 and its β2 subunit, but not Nav1.6 or β1, are clustered in developing central nervous system nodes and that clustering of Nav1.2 and Nav1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Nav1.2 α and β2 subunits along central nervous system axons in...
From the intracellularly recorded responses to small, rapidly flashed spots, we have quantitatively mapped the receptive fields of simple cells in the cat visual cortex. We then applied these maps to a feedforward model of orientation selectivity. Both the preferred orientation and the width of orientation tuning of the responses to oriented stimuli were well predicted by the model. Where tested,...
Neuropeptides are ubiquitous transmitters that have been implicated in a wide variety of physiological and pathological conditions, and it is important to understand the processes that control their secretion. We have developed a technique that measures neuropeptide secretion with high temporal resolution. This method involves placing an electrophysiological “tag” in a neuropeptide prohormone. The...
Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel α subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Nav1.2 is localized to the unmyelinated...
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