Exposure of young children to general anesthesia (GA) is common in medicine; however, emerging data suggest that early postnatal exposure to GA may be detrimental to brain development, resulting in long- term cognitive impairments. Older literature also suggested that in utero exposure of rodents to GA causes cognitive impairments in the first- as well as the second-generation offspring never exposed to GA. Thus, we must consider the general hypothesis that transient exposure to GA during critical stages of brain development causes epigenetic changes in chromatin and DNA with deleterious effects on transcription of target genes crucial for proper synapse formation and cognitive development. Our long-term goals are to understand how GA modulates transcriptional machinery of developing neurons and to establish any links between GA-induced epigenetic modulations and long-term memory deficit. The rationale is that this understanding will enable pharmacological targeting of critical steps in gene transcription aimed to prevent GA-induced impairment of synaptogenesis and memory/learning deficits. We will use in vivo and in vitro rat models of GA-induced developmental neurotoxicity to address the specific hypothesis that GA decreases histone acetylase (HAT) activity of cAMP-responsive element binding protein (CREB) Binding Protein (CBP) and thereby reduces transcription of target genes that regulate the development of neuronal morphology, function and long-term memory. Specific Aim #1: Determine how GA alters histone acetylation and thereby the activity of a set of transcription factors (CREB, c-FOS) that target genes contributing to long-term memory storage and consolidation (e.g. BDNF, MECP2, Cdk-5). Preliminary data suggest that GA significantly decreases CBP content and global histone 3 (H3) acetylation, the epigenetic modulations that inhibit transcriptional activation. We will examine GA effects on total HAT and on histone deacetylase (HDAC) activities via ELISA and on specific HAT activity of CBP after immunoprecipitation; and follow acetylation at specific histone sites with antibodies to H3K14, H2BK12 and H4K8. Expression and phosphorylation of cFos and CREB will be assessed, and activation of target genes will be assayed via DNA methylation, targeted assessment of histone acetylation in their promoters, and mRNA production. The dependence of GA-induced changes in histone acetylation will be assessed by determining whether isoform-specific HDAC inhibitors or isoform-specific HDAC siRNA can reverse them. Specific Aim #2: Use specific HDAC inhibitors or HDAC siRNA to determine whether GA- induced epigenetic changes in histone acetylation and transcriptional regulation of CREB, c-FOS, and target genes are linked to GA-induced impairment of neuronal morphology, dendritic arborization, synapse formation, synaptic plasticity and memory development. We will attempt to mimic the GA effect in neuronal cultures and slices by using CBP siRNA to impair its HAT and transcriptional activity and CREB siRNA to impair CBP:CREB interaction.