tetrodotoxin and Teratocarcinoma

Understanding the structural and functional development of neurons in networks has a high impact to estimate the potentials for restorative therapies. Neurons derived from the human NT2 cell line (hNT) formed networks with a clustered neuritic architecture in vitro, whereas primary dissociated embryonic rat cortical neurons (Cx) displayed a more homogenous cell assembly. Spontaneous spikes of both cell types were recorded on microelectrode arrays within 2 weeks after seeding, but hNT showed a mostly uncorrelated firing pattern in contrast to Cx with highly synchronized bursting. hNT neurons were less sensitive to TTX (IC50 = 5.7 +/- 0.1 nM vs. IC50 = 1.1 +/- 0.2 nM), magnesium (IC50 = 1.83 +/- 0.01 mM vs. IC50 = 0.161 +/- 0.023 mM), and APV (IC50 > 100 microM vs. IC50 = 18 microM). We conclude that embryonic cortical neurons and hNT neurons have different network properties. This should be carefully considered before hNT neurons are used in therapeutic approaches, e g., central nervous system (CNS) grafting.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Axons; Biomarkers; Brain Tissue Transplantation; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Fetus; Humans; Magnesium; Microelectrodes; Nerve Net; Nerve Tissue Proteins; Neural Pathways; Neurons; Rats; Teratocarcinoma; Tetrodotoxin

We report that human hNT cells display neuron-like calcium channel activation. Patch-clamp experiments show that exposure of hNT cells to the Alzheimer-related amyloid peptide beta AP(25-35) induces large and irreversible inward calcium currents at -80 mV in whole cell mode, with a linear current-voltage relationship. This behavior is suggestive of ionophore formation. An analogous peptide with scrambled sequence has no effect. These ionophore effects by the beta AP(25-35) peptide, the first report in a human cell-line, are very rapid effects. The currents are large and stable, and are blocked by Al3+ but not by Cd2+. Filtration removes a peptide aggregate from the amyloid peptide beta AP(25-35) solution and thereby abolishes the inward current. The residual soluble peptide has no effect. These data suggest that the initial step of the neurotoxic effect of beta AP(25-35) may be due to the insertion of the aggregated peptide into the cellular membrane as a Ca2(+)-carrying ionophore. The relevance of calcium-mediated cell death, especially in Alzheimer's disease, is discussed.

Topics: Aluminum; Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Calcium; Electric Conductivity; Humans; Ion Channel Gating; Ionophores; Molecular Sequence Data; Neurons; Patch-Clamp Techniques; Teratocarcinoma; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Tumor Cells, Cultured

NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na+ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, evoked whole-cell currents. In addition, gamma-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development.

Topics: Bicuculline; Cell Differentiation; Electric Conductivity; Evoked Potentials; gamma-Aminobutyric Acid; Humans; Kainic Acid; Membrane Potentials; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Receptors, GABA-A; Sodium Channels; Teratocarcinoma; Tetrodotoxin; Tretinoin; Tumor Cells, Cultured