Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures
Section snippets
Mature hippocampal-entorhinal cortex slice preparation
Organotypic slices were prepared from 25 to 30 day old male Wistar rats (Institute breeding stock; SHOE, Magdeburg, Germany) as described previously (Leutgeb et al., 2003). All experiments conformed to institutional, state (Land Sachsen-Anhalt) and international guidelines for the ethical use of animals. Efforts were made to minimize the number of animals used as well as any suffering. Slices were incubated overnight at 34 °C in a humidified carbogen atmosphere (95% O2/5% CO2), and then
Forskolin bath application induces uniform CREB phosphorylation
It was first tested whether changes in CREB phosphorylation could be reliably detected at a single cell resolution. Bath application of forskolin, an activator of the PKA cascade, has been shown to result in an increase in CREB phosphorylation (Kanterewicz et al., 2000). Analysis of forskolin-induced changes in CREB phosphorylation was performed using mature hippocampal-entorhinal cortex slices after 12 DIV. Slices were incubated in forskolin (20 μM) for various time periods (0, 5, 30, 60, 120,
Discussion
The acute hippocampal slice has been a preferred model for the investigation of LTP. It has been shown that the routine incubation of acute slices can result in the up-regulation of immediate early-genes, whose transcription depends on CREB activation, presumably as a result of hypoxic injury and apoptosis (Zhou et al., 1995). Moreover, CREB can be specifically phosphorylated during the activation of neuronal cell death and apoptosis (Vyas et al., 2002) as well as in the processes of neuronal
Acknowledgments
We thank Dr. S. Leutgeb for his valuable scientific discussions and critical review of the manuscript. This research was supported by the Volkswagen Foundation grant I/78 555 to Dr. T. Behnisch.
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