Targeting green fluorescent protein to dendritic membrane in central neurons.
Dendritic and axonal processes are input and output sites, respectively, of neuronal information, and detailed visualization of these processes may be indispensable for elucidating the neuronal circuits and revealing the principles of neuronal functions. To establish a method for completely visualizing dendritic processes, we first developed green fluorescent protein (GFP)-based proteins and, by using lentivirus with a neuron-specific promoter, examined whether or not the protein fully visualized the dendritic processes of infected neurons. When GFP with a palmitoylation (palGFP) or myristoylation/palmitoylation site (myrGFP) was expressed in rat brain with lentiviruses, myrGFP labeled dendritic membrane better than palGFP. Subsequently, dendrite-targeting efficiencies of three basolateral membrane-sorting and three putative dendrite-targeting domains, which were attached to myrGFP C-terminus, were examined in striatonigral GABAergic and corticothalamic glutamatergic neurons, and in cultured cortical neurons. Of the six domains, C-terminal cytoplasmic domain of low density lipoprotein receptor (LDLRct) was most efficient in targeting the protein to dendrites, showing 8.5-15-fold higher efficiency in striatonigral neurons compared with myrGFP. Finally, dendritic membrane-targeting potency of myrGFP-LDLRct was confirmed in transgenic mice using Thy1 or Gad1 expression cassette. Thus, myrGFP-LDLRct is an excellent synthetic protein for dendritic visualization, and may be a useful tool for the morphological analysis of neuronal circuits.
Kameda
Hiroshi
H
Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Furuta
Takahiro
T
Matsuda
Wakoto
W
Ohira
Koji
K
Nakamura
Kouichi
K
Hioki
Hiroyuki
H
Kaneko
Takeshi
T
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D003712:Dendrites / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011973:Receptors, LDL / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000502:physiology
D011973:Receptors, LDL; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid; D004247:DNA