Microinjection of mRNA 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 technique for modifying gene expression in identified motoneurons of the crayfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RNAs. mRNA suitable for injection was transcribed in vitro from cDNA templates cloned into a plasmid, pSEM. Initially, mRNAs encoding green fluorescent 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 expressed. Measurements of GFP fluorescence increase indicated that GFP mRNA was stable and translated into protein for at least 3 days postinjection. We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synaptic physiology. The mRNA injection procedure did not appear to directly influence synaptic physiology based on the results of the AASP-168 and GFP injections. Injection of mRNA encoding rat synapsin Ia resulted in a significant increase in peak excitatory postsynaptic potential (EPSP) amplitude during repetitive stimulation. These data are consistent with previous studies that have shown that synapsin deficiency reduces synaptic vesicle numbers. The translation of mRNAs with diverse functions and species of origin suggests that this approach will prove useful for studying the function of a wide variety of endogenous and exogenous genes in identified neurons.
Dearborn
R E
RE
Department of Biological Sciences, University at Albany, State University of New York 12222, USA.
Szaro
B G
BG
Lnenicka
G A
GA
D000818:Animals; D003400:Astacoidea / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials; D017930:Genes, Reporter; D005815:Genetic Code; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D009046:Motor Neurons / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics; D018377:Neurotransmitter Agents / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013569:Synapses / Q000502:physiology; D016704:Synapsins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D012333:RNA, Messenger; D016704:Synapsins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase