Biomedical subjects

H Nadeau

Publications and source records attributed to H Nadeau.

14 recordsLinked to original sources

PubMed2002

NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons.

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

Animals↗

PubMed2000

Two-compartment model for whole-cell data analysis and transient compensation.

Recording and analysis of neuronal patch-clamp data involve many assumptions about membrane properties and cell morphology. Some of these assumptions introduce large errors or oversimplifications into the results. In particular, dendritic branching with high intracellular resistance leads to difficulty with capacitance calculation and transient subtraction, and may significantly distort measured currents. A two-compartment model, presented in detail here, provides a simple method of reducing many of these problems for the relatively simple case of cultured neurons studied with whole-cell patch electrodes. Some passive membrane properties may be accurately calculated, and the results may be used to correct recorded currents for resulting series resistance, intracellular resistance, and capacitive transient errors. The model may be tailored to particular cell types or experimental conditions. Programs to implement the algorithms are available from http://www.its.caltech.edu/ approximately nadeau/Rscomp.html.

Animals↗

PubMed2000

ROMK1 (Kir1.1) causes apoptosis and chronic silencing of hippocampal neurons.

Lentiviral vectors were constructed to express the weakly rectifying kidney K(+) channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2-4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K(+) loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at -144 mV in 5.4 mM external K(+) blockable by 500 microM Ba(2+), ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba(2+)-induced depolarization of 7 +/- 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 +/- 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K(+) has implications for the use of K(+) channels in gene therapy.

Action Potentials↗

PubMed1996