The goal of this R21 exploratory grant is to systematically develop the use of RNA interference (RNAi) in Aplysia neurons. Electrophysiological and behavioral studies in Aplysia have delineated the circuitry mediating simple forms of learning and memory in the animal. Cultured sensory-motor neurons from Aplysia have provided a model system for elucidating many of the molecular and cell biological mechanisms underlying learning-related synaptic plasticity. These mechanisms have been found to be generalizable to learning-related neuronal plasticity across species. While Aplysia offers many experimental advantages for cell biological and electrophysiological studies, it has not been suitable for genetic analyses. RNAi technology promises to transform Aplysia into a system in which genetic, behavioral, electrophysiological and cell biological analyses can be performed both in the animal and at the level of single cells and synapses. The experiments outlined in this proposal are aimed at developing methodologies for the use of RNAi in Aplysia. We will focus on investigating and optimizing 1) the type of RNA used for RNAi-long double stranded RNA (dsRNA) or small interfering RNAs (siRNAs)-- and 2) the method of delivery of the RNAi. To do this, we will target four endogenous Aplysia genes as well as exogenously overexpressed destabilized eGFP. We will determine whether the RNAi effectively silences target genes and whether or not this silencing is specific to the target gene. In addition, our experiments will identify the most efficient means of delivering RNA for RNAi and the best techniques for assaying the efficacy and specificity of RNAi-mediated gene silencing in Aplysia neurons. The results of the proposed experiments will be invaluable to researchers working in the Aplysia model system. From a broader perspective, the ability to use RNAi in Aplysia will generate valuable information about the molecular mechanisms underlying synapse formation, synaptic transmission and synaptic plasticity. This information likely will lead to the identification of potential therapeutic targets for the many neurological and psychiatric diseases in which these fundamental processes are perturbed. We propose to improve methods to study the molecular basis of learning and memory. The technologies we propose to develop will identify genes that are required for learning and in so doing will lead to potential therapies for the many diseases in which learning and memory are altered. Such diseases include mental retardation, age-related memory loss, Alzheimer's disease, drug addiction as well as many neuropsychiatric diseases. ? ?
| Zhao, Yali; Wang, Dan O; Martin, Kelsey C (2009) Preparation of Aplysia sensory-motor neuronal cell cultures. J Vis Exp : |
