Inhibitory neuron‐specific Cre‐dependent red fluorescent labeling using VGAT BAC‐based transgenic mouse lines with identified transgene integration sites

Abstract

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.

The structural and electrophysiological properties of progesterone receptor expressing neurons vary along the anterior-posterior axis of the ventromedial hypothalamus and undergo local changes across the reproductive cycle

“…In fact, the absence of GABAergic somas has been used to delineate the boundaries of the VMH (Jarvie & Hentges, 2012). Taking advantage of mice expressing the fluorescent reporter tdTomato under the promoter of the vesicular transporter of GABA (VGat-tdTomato) (Kaneko et al, 2018) we observed, as expected, lower signal intensity inside the boundaries of the VMH (Fig. 6A) that was consistent across the AP regions of the VMH.…”

GABAergic neuron-specific whole-brain transduction by AAV-PHP.B incorporated with a new GAD65 promoter

“…Wild-type and VGAT-tdTomato mice from C57BL/6 backgrounds [18] were used in this study. All procedures for the care and treatment of animals were performed according to the Japanese Act on the Welfare and Management of Animals and the Guidelines for Proper Conduct of Animal Experiments issued by the Science Council of Japan.…”

“…2a). Because the VGAT-tdTomato mice expressed tdTomato specifically in inhibitory interneurons throughout the central nervous system (CNS) [18], we determined whether the transduced (GFP-expressing) cells were inhibitory GABAergic neurons without immunohistochemistry. The mice were sacrificed 3 weeks after viral injection.…”

GABAergic Neuron-Specific Whole-Brain Transduction by AAV-PHP.eB Incorporated with a New GAD65 Promoter

“…Wild-type and VGAT-tdTomato mice of C57BL/6 backgrounds [18] were used in this study. All procedures for the care and treatment of animals were performed according to the Japanese Act on the Welfare and…”

“…To examine whether the mGAD65 promoter works exclusively in inhibitory neurons, we injected VGAT-tdTomato mice with AAV-PHP.B-expressing GFP under the control of the mGAD65 promoter or mDlx enhancer (100 µL, 5.0 × 10 13 viral genome/ml) intravenously, which expressed tdTomato speci cally in inhibitory interneurons throughout the central nervous system (CNS) [18] ( Fig. 2A).…”