A versatile microporation technique for the transfection of cultured CNS neurons.

The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simple and efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique that efficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Microporation allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higher percentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higher levels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporated neurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability to produce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein (GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum. The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherent neurons in a 3 mm2 surface area while requiring as little as 1 microl of the material to be introduced. We conclude that the microporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.

A post-docking role for synaptobrevin in synaptic vesicle fusion.

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%-68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.