Targeting green fluorescent protein to dendritic membrane in central neurons

Abstract

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.

Introduction

Since dendrites and axons of neurons are input and output sites, respectively, of information, detailed visualization of these processes and their connections may be indispensable for elucidating the basic design of neuronal circuits and thereby revealing the principles of neuronal functions. There are several methods for visualizing the axons of a functional group of neurons, such as immunocytochemical staining. For example, parvalbumin and somatostatin are selectively produced by different subsets of GABAergic interneurons in the cerebral cortex, and their immunoreactivities are observed not only in cell bodies but also in axon fibers and terminals (Bennett-Clarke et al., 1980, DeFelipe et al., 1989, van Brederode et al., 1991). By contrast, the distal portions of dendrites or dendritic spines are not visualized well in immunocytochemistry with a few exceptions such as immunoreactivities for NK1 receptor (NK1R) (Nakaya et al., 1994) and telencephalin (TLC) (Mitsui et al., 2005). Only the old method of Golgi stain and intracellular staining technique completely visualizes dendritic processes of CNS neurons. However, the Golgi method allows us to label neurons only in a non-selective manner, and the intracellular staining technique is usually applied for labeling of a small number of neurons and unsuitable for entire visualization of a functional group of neurons. Thus, a novel method for fully visualizing the dendrites of a functional neuron group has long been desired in the field of neuroscience.

Recently, gene-technological devices such as viral vectors, which introduce a gene for enhanced green fluorescent protein (GFP) attached with a plasma membrane-targeting signal, have been developed for labeling of axonal and dendritic contours (Moriyoshi et al., 1996, Tamamaki et al., 2000, Furuta et al., 2001). If a more sensitive and selective tool for labeling dendrites in a Golgi-stain-like manner is developed, the input sites of a functional neuron group can be visualized by combining the tool with techniques generating transgenic animals. Here we first re-examined GFPs with a plasma membrane-targeting signal by using replication-deficient lentivirus with a neuron-specific promoter (Hioki et al., 2007). Lentiviral vectors were used because genes delivered by lentivirus are incorporated into host genome, and thereby the vector might be a good precursory test system for gene expression in transgenic animals. Second, dendritic membrane-targeted GFP was developed by fusing the plasma membrane-targeted GFP with basolateral sorting signals of polarized epithelial cells and putative dendrite-targeting signals of neurons. We finally applied GFP fused with the best dendritic membrane-targeting signal to transgenic animal generation and succeeded in entirely visualizing the dendrites of a neuron group determined by gene expression.