Development of lentiviral vectors for efficient glutamatergic-selective gene expression in cultured hippocampal neurons

Yoshihiro Egashira,Yuchio Yanagawa,Yasunori Mori,Shigeo Takamori

doi:10.1038/s41598-018-33509-5

Yoshihiro Egashira, Yuchio Yanagawa + Show 2 more

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https://doi.org/10.1038/s41598-018-33509-5

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Journal: Scientific Reports	Publication Date: Oct 11, 2018
Citations: 12	License type: open-access

Affiliation: Doshisha University, Gunma University

Abstract

Targeting gene expression to a particular subset of neurons helps study the cellular function of the nervous system. Although neuron-specific promoters, such as the synapsin I promoter and the α-CaMKII promoter, are known to exhibit selectivity for excitatory glutamatergic neurons in vivo, the cell type-specificity of these promoters has not been thoroughly tested in culture preparations. Here, by using hippocampal culture preparation from the VGAT-Venus transgenic mice, we examined the ability of five putative promoter sequences of glutamatergic-selective markers including synapsin I, α-CaMKII, the vesicular glutamate transporter 1 (VGLUT1), Dock10 and Prox1. Among these, a genomic fragment containing a 2.1 kb segment upstream of the translation start site (TSS) of the VGLUT1 implemented in a lentiviral vector with the Tet-Off inducible system achieved the highest preferential gene expression in glutamatergic neurons. Analysis of various lengths of the VGLUT1 promoter regions identified a segment between −2.1 kb and −1.4 kb from the TSS as a responsible element for the glutamatergic selectivity. Consistently, expression of channelrhodopsin under this promoter sequence allowed for selective light-evoked activation of excitatory neurons. Thus, the lentiviral system carrying the VGLUT1 promoter fragment can be used to effectively target exogenous gene expression to excitatory glutamatergic neurons in cultures.

Highlights

The mammalian central nervous system (CNS) consists of two major classes of neurons, glutamatergic excitatory neurons and GABAergic inhibitory neurons
Lentiviral vectors equipped with the human synapsin I promoter and the mouse α-CaMKII promoter showed selectivity toward glutamatergic neuron in the brain in vivo[5,6,7], we found that these promoters caused reporter expression in a substantial fraction of GABAergic neurons when used in the cultured preparation (Fig. 1b)
dentate gyrus granule cells (DGGCs) represent a limited fraction of hippocampal glutamatergic neurons, targeted gene expression in DGGCs in a culture preparation would be profitable for another line of studies because of their characteristic cellular functions, such as presynaptic plasticity in their mossy fiber boutons[20]

Summary

Introduction

The mammalian central nervous system (CNS) consists of two major classes of neurons, glutamatergic excitatory neurons and GABAergic inhibitory neurons. Attempts to establish an experimental tool for the genetic manipulation of neuronal cells in vivo revealed that lentiviral vectors equipped with either 0.4–1.1 kb fragments of the synapsin I promoter region or a ~1.3 kb fragment of the α-CaMKII promoter region effectively target gene expression to cortical neurons in the intact rodent brain[5,6,7]. It appears that both promoters show a strong preference for excitatory neurons, with weak www.nature.com/scientificreports/. Several groups have exploited the Tet-Off inducible system in combination with established lentiviral vectors resulting in highly efficient transgene expression in a neuron-specific manner[10,11,12]

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