Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 lM) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter.Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

We present evidence that the pond snail Lymnaea stagnalis is capable of aquisition and extensive retention of an appetitively reinforced feeding response after only a single training trial. Food-deprived snails presented with a single pairing of a phagostimulant (a mixture of sucrose and casein digest) and a novel, non-food chemostimulus (amyl acetate) subsequently made feeding responses to the amyl acetate and retained the association for at least 19 days. This demonstration of one-trial, non-aversive classical conditioning enhances the utility of Lymnaea stagnalis as a model system for the detailed analysis of neural mechanisms underlying plasticity.

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A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases selectively modulate different aspects of neurite development. The university of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involved in neurite development. Finally, our data indicate that some superficially similar characteristics of neuronal differentiation, such as neurite initiation and branching, may be controlled by quite different molecular mechanisms.