MICROINJECTION OF MESSENGER-RNA ENCODING RAT SYNAPSIN IA ALTERS SYNAPTIC PHYSIOLOGY IN IDENTIFIED MOTONEURONS OF THE CRAYFISH, PROCAMBARUS-CLARKII

Studies of identified neurons have made important contributions to our understanding of cellular neurophysiology. We have developed a techni que for modifying gene expression in identified motoneurons of the cra yfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RN As, mRNA suitable for injection was transcribed in vitro from cDNA tem plates cloned into a plasmid, pSEM, Initially, mRNAs encoding green fl uorescent protein (GFP) and beta-galactosidase were injected into the soma of the motor giant neuron (MoG) to determine whether these mRNAs could be successfully translated into protein. Both proteins were expr essed. Measurements of GFP fluorescence increase indicated that GFP mR NA was stable and translated into protein for at least 3 days postinje ction, We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synap tic physiology. The mRNA injection procedure did not appear to directl y influence synaptic physiology based on the results of the AASP-168 a nd GFP injections. Injection of mRNA encoding rat synapsin Is resulted in a significant increase in peak excitatory postsynaptic potential ( EPSP) amplitude during repetitive stimulation. These data are consiste nt with previous studies that have shown that synapsin deficiency redu ces synaptic vesicle numbers. The translation of mRNAs with diverse fu nctions and species of origin suggests that this approach will prove u seful for studying the function of a nide variety of endogenous and ex ogenous genes in identified neurons. (C) 1998 John Wiley Sr Sons, Inc.