Elsevier

Neuroscience

Volume 131, Issue 3, 2005, Pages 601-610
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2004.12.002Get rights and content

Abstract

Phosphorylation of the transcription factor cyclic AMP (cAMP)-response element-binding protein (CREB) has been implicated in long-term synaptic plasticity and memory, and its activation has been proposed to be required for the maintenance of long-term potentiation (LTP). The previously described temporal dynamics of CREB phosphorylation during the maintenance of LTP showed differences between experimental models. In the present study the level of CREB phosphorylation was evaluated in organotypic hippocampal slices from young adult rats (P25-30) after long-lasting LTP was induced. Immunohistochemistry and confocal imaging were used to determine the ratio between non-phosphorylated and phosphorylated CREB at a single cell resolution, revealing not only the temporal dynamics but also the extent of CREB phosphorylation. The activation of CREB after LTP-induction was compared with cAMP-activation after bath application of forskolin. An increase in cAMP by forskolin resulted in a persistent, uniform increase of the phosphorylated CREB (pCREB/CREB immunofluorescence ratio) in all hippocampal principal neurons. In contrast, the induction of long-lasting LTP in CA1 was accompanied by a local increase in the pCREB/CREB ratio. Both CREB activation and LTP induction in mature cultured slices required N-methyl-d-aspartate (NMDA) receptor activation. CREB phosphorylation continued to increase for 4 h during LTP maintenance. This sustained activation is in contrast to previous observations in acutely prepared slices and supports the hypothesis that CREB plays an important role during the late phases of LTP.

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.

References (30)

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