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Pathways to CREB activation: Creb activation downstream of ion channels

Numerous in vivo studies have demonstrated that psychostimulant drugs such as amphetamine and cocaine can induce the expression of the immediate early gene c-fos in striatal neurons via the activation of D1 dopamine receptors. NMDA receptor activation is also known to induce c-fos in the striatum. A primary striatal neuronal culture preparation was used to examine the mechanisms whereby these stimuli lead to changes in gene expression. Direct application of NMDA to striatal cells in culture causes a rapid increase in the expression of c-fos, as well as an increase in the phosphorylation of the transcription factor CRE binding protein (CREB). This is prevented by NMDA receptor antagonists, and requires extracellular calcium, but does not involve L-type calcium channels. The induction of c-fos and CREB phosphorylation following NMDA are unaffected by inhibition of protein kinase C, tyrosine kinases or nitric oxide synthase. However, the response to NMDA is blocked by KN62, a selective inhibitor of calcium/calmodulin-dependent protein kinase. The application of the D1 agonist SKF 38393, or the direct stimulation of adenylyl cyclase with forskolin, also results in the phosphorylation of CREB and the induction of c-fos in striatal neurons. These effects are blocked by the protein kinase A inhibitor H89. These observations are consistent with the hypothesis that calcium/calmodulin-dependent phosphorylation of CREB induced by NMDA, or cAMP-dependent phosphorylation of CREB induced by D1 agonists, underlie the induction of c-fos seen following activation of these receptors in striatal neurons (Das, 1997).

The second messenger pathways linking receptor activation at the membrane to changes in the nucleus are just beginning to be unraveled in neurons. The work presented here attempts to identify in striatal neurons the pathways that mediate cAMP response element-binding protein (CREB) phosphorylation and gene expression in response to NMDA receptor activation. The phosphorylation of the transcription factor CREB, the expression of the immediate early gene c-fos, and the induction of a transfected reporter gene under the transcriptional control of CREB after stimulation of ionotropic glutamate receptors were investigated. Neither AMPA/kainate receptors nor NMDA receptors are able to independently stimulate a second messenger pathway that leads to CREB phosphorylation or c-fos gene expression. Instead, a consecutive pathway from AMPA/kainate receptors to NMDA receptors and from NMDA receptors to L-type Ca2+ channels is seen. AMPA/kainate receptors are involved in relieving the Mg2+ block of NMDA receptors, and NMDA receptors trigger the opening of L-type Ca2+ channels. The second messenger pathway that activates CREB phosphorylation and c-fos gene expression is likely activated by Ca2+ entry through L-type Ca2+ channels. It is concluded that in primary striatal neurons glutamate-mediated signal transduction is dependent on functional L-type Ca2+ channels (Rajadhyaksha, 1999).

AMPA/kainate receptor channels open after interaction with glutamate and permit Na+ entry at the synapse. The resulting local depolarization removes the Mg2+ block of the NMDA receptor, which permits the NMDA receptor to respond to extracellular glutamate and glycine. Opening of the NMDA receptor channel causes Na+ and Ca2+ influx. Unlike the AMPA/kainate receptor channel that desensitizes rapidly, NMDA receptor channels have long opening times. Therefore, NMDA receptors can trigger the opening of L-type Ca2+ channels that open during strong depolarization. The activation of L-type Ca2+ channels promotes Ca2+ entry along the dendrites and at the cell body. Second messengers activated by Ca2+ translocate to the nucleus and phosphorylate CREB. The results presented in this paper suggest an important role for L-type Ca2+ channels in neuroplasticity of the striatum and confirm previous reports about the involvement of L-type Ca2+ channels in NMDA-mediated plasticity and toxicity. Under the experimental conditions described in this study, NMDA receptors initiate a signal transduction pathway but do not initiate a significant intraneuronal second messenger pathway, either alone or together with AMPA/kainate receptors. Depolarization of L-type Ca2+ channels plays a crucial role in the activation of an intraneuronal second messenger pathway (Rajadhyaksha, 1999).

Although the supportive role of AMPA/kainate receptors for NMDA receptors is in agreement with previous findings in hippocampal culture, other findings differ. In hippocampal cultures NMDA receptors and L-type Ca2+ channels seem to contribute to independent, parallel pathways rather than to the same pathway. Like in hippocampal cultures, L-type Ca2+ channels in the striatum activate the CRE and function independently of NMDA receptors. But although a direct pathway from NMDA receptors to the SRE in the striatum cannot be excluded, this pathway in itself is not enough to mediate c-fos gene expression. This difference may be attributed to intrinsic differences between both types of neurons or to the different neurotransmitters released in either culture. Hippocampal neurons are mostly glutamatergic and express very high levels of glutamate receptors. Striatal cultures are primarily GABAergic and express much lower levels of glutamate receptors. Because neurons in culture synapse onto each other, hippocampal neurons excite each other after activation, whereas GABA in striatal neurons, dependent on the level of maturity, may be excitatory or inhibitory. To avoid trans-synaptic effects in hippocampal cultures, Na+ channels are often blocked with TTX. Thus, there are fundamental differences in glutamate-mediated gene expression in neurons of both brain areas (Rajadhyaksha, 1999).

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CrebB-17A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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