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SummaryPurpose-We assayed the effects of rapamycin, an immunomodulatory agent known to inhibit the activity of the mammalian target of rapamycin (mTOR) cascade, on candidate gene expression and single unit firing properties in cultured rat hippocampal neurons as a strategy to define the effects of rapamycin on neuronal gene transcription and excitability.Methods-Rapamycin was added (100 nM) to cultured hippocampal neurons on days 3 and 14. Neuronal somatic size and dendritic length were assayed by immunohistochemistry and digital imaging. Radiolabeled mRNA was amplified from single hippocampal pyramidal neurons and used to probe cDNA arrays containing over 100 distinct candidate genes including cytoskeletal element, growth factor, transcription factor, neurotransmitter, and ion channel genes. In addition, the effects of rapamycin (200 nM) on spontaneous neuronal activity and voltage-dependent currents was assessed.Results-There were no effects of rapamycin on cell size or dendrite length. Rapamycin altered expression of distinct mRNAs in each gene family on days 3 and 14 in culture. Single unit recordings from neurons exposed to rapamycin exhibited no change from baseline. When spontaneous activity was increased by blocking GABA-mediated inhibition with bicuculline, a fraction of the neurons exhibited a decreased duration of spontaneous bursts and a decrease in synaptic inputs. Rapamycin did not appear to alter voltage dependent Na+ or K+ currents underlying action potentials.Conclusions-These data demonstrate that rapamycin does not produce neurotoxicity nor alter dendritic growth and complexity in vitro and does not significantly alter voltage gated sodium and potassium currents. Rapamycin does affect neuronal gene transcription in vitro. Use of rapamycin in clinical trials for patients with tuberous sclerosis complex should involve vigilance for possible effects on seizure frequency and neurocognitive function.