Cellular neuroscience(R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus
Section snippets
Ethical approval
All animal protocols were approved by and conformed to the French Public Health Service policy and INSERM guidelines on the use of laboratory animals. All experiments were performed according to the international guidelines on the ethical issues. All efforts were made to minimize animal suffering and reduce the number of animals used.
Primary cultures of rat hippocampal neurons
Hippocampal neurons from newborn rats were dissociated using trypsin and plated on coverslips coated with poly-l-lysine at a density of 70,000 cells cm−2 in
(R)-roscovitine generates a tonic current mediated by GABAARs
Bath application of 10 μM (R)-roscovitine produced a positive shift of the current baseline (67.4±35.5 pA, n=12, P<0.01) in cultures older then 13 days in vitro (DIV) (Fig. 1A, upper trace; Vh=40 mV, cesium gluconate solution) and had no effect in younger cultures (data not shown). Current baseline returned to the initial level a few seconds after washout of (R)-roscovitine (Fig. 1A, lower trace). To explore the voltage dependence of (R)-roscovitine-induced current, the compound was applied for
Discussion
The principal finding of this study is that (R)-roscovitine enhances GABA-mediated tonic current in the hippocampal cultured neurons and hippocampal slices. (R)-roscovitine had no effect in cultures younger than 13DIV. This result is similar to that reported recently by Wu and Richerson, 2006, where they observed a strong age dependence of tonic inhibition in hippocampal culture. The effect of (R)-roscovitine is not sensitive to extracellular calcium concentration, requires a functioning of
Acknowledgments
We thank Dr. L. Meijer for providing the samples of roscovitines, Dr. I. Medyna and Dr. T. Harkany for help with immunostaining and confocal microscopy analysis. We are grateful to Dr. G. Richerson for helpful comments on an early version of this paper. We thank also Dr. D. A. Turner for critical reading of the manuscript. Supported by a European Community grant LSHB-CT-2004-503467 A.I., R.T, and Y.Z.
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