Transfecting and Transducing Neurons with Synthetic Nucleic Acids and Biologically Active Macromolecules - PubMed (original) (raw)

A major challenge in neurobiology is to better understand the physiological role of the protein–protein interactions and post-translational modifications important for regulating the activity and plasticity of synapses. A significant effort has been undertaken over the past 10 years to identify the protein components of the post-synaptic domain and the protein machinery important for regulating the activity of the neurotransmitter receptors that mediate the electrophysiological responses responsible for synaptic activity. Of significant contribution to this effort has been the systematic identification of the protein constituents and protein–protein interactions of the synapse, using proteomic and yeast two-hybrid approaches and the identification and characterization of the signalling pathways, enzymes and synaptic substrates of particular receptor post-translational modifications (such as phosphorylation, palmitoylation, ubiquitination and nitrosylation) important in the regulation of neurotransmitter receptor activity and synaptic plasticity [1–5].

Essential to the functional characterization of the molecular machinery important for regulating synapse development, synaptic transmission and synaptic plasticity has been the development of methods for altering the constituents of a neuron either genetically or by changing the activity of a protein of interest. Although homologous recombination in embryonic stem (ES) cells is now a standard method to mutate the germ line of mice and hence interfere with the activity of a particular gene of interest [6], this approach still has several limitations. These include the high costs of producing and maintaining transgenic animals, complications due to embryonic lethality and the potential consequences on nervous-system development of introducing into the animal a particular mutation. Other potential limitations include genetic compensation, spatial and temporal specificity and the restriction of working with mouse tissues. As a consequence, a significant effort has been directed toward developing and improving methods for introducing biological agents (including DNA, proteins, antibodies and biologically active or protein–protein interaction-blocking peptides) into either single or large populations of neurons. Here, we consider some techniques that have emerged for transfecting and transducing neurons with DNA and biologically active macromolecules. We focus, in particular, on the use of these approaches for studying the molecular mechanisms important for regulating the activity and plasticity of synapses and the functional regulation of the key ionotropic glutamate receptors (NMDA receptors, AMPA receptors and kainate receptors) and inhibitory ligand-gated ion channels (GABAA and glycine receptors) at excitatory and inhibitory synapses, respectively [1,7–10].