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

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The brain orchestrates animal behaviors and the function of the brain itself and consists of a tremendous number of neurons and non‐neuronal cell types. Neurons control brain function through the formation of highly organized neural circuits of interconnected excitatory and inhibitory neurons, which consist of GABAergic and glycinergic neurons in vertebrates. Although inhibitory neurons represent a minor proportion of neurons (e.g., 10–20% in the rodent neocortex and 20–30% in the primate neocortex), their numbers, distribution, dendritic and axonal morphologies, and connections are crucial for shaping and regulating the dynamics of the entire network (Achim, Salminen, & Partanen, 2014; Defelipe, 2011; Isaacson & Scanziani, 2011; Pouille & Scanziani, 2001). Abnormalities in inhibitory neurons, which lead to an excitatory/inhibitory imbalance, are implicated in aging‐related impairments (Crosson et al., 2015; Stanley, Fadel, & Mott, 2012) and several developmental disorders, including epilepsy (Armijo, Valdizán, De Las Cuevas, & Cuadrado, 2002), autism (Gaetz et al., 2014; Lawrence, Kemper, Bauman, & Blatt, 2010; Mariani et al., 2015; Rojas, Singel, Steinmetz, Hepburn, & Brown, 2014; Rubenstein & Merzenich, 2003), and schizophrenia (Lewis, 2000). Thus, an understanding of the molecular mechanism underlying the numbers, distribution, axonal and dendritic morphologies, and connections of inhibitory neurons might reveal some causes of and potential therapeutic approaches to these complex disorders, and fluorescent labeling of inhibitory neurons has greatly facilitated studies aimed at understanding the roles of inhibitory neurons in the brain.
A number of transgenic (Tg)/knock‐in (KI) mouse lines that express fluorescent proteins in defined subsets of neurons, including inhibitory neurons, have been generated and have provided powerful tools to study neurons of interest (Tsien, 2003). One limitation of this methodology, however, is that the majority of the Tg/KI mice use green fluorescent protein/yellow fluorescent protein (GFP/YFP) as the fluorescent probe. Because of the limited availability of lines using red fluorescent protein (RFP), the simultaneous visualization of inhibitory neurons and other cell populations in the same preparation has been difficult. Our laboratory and other groups have previously generated Tg/KI mouse lines that express GFP/YFP in inhibitory neurons (Chattopadhyaya et al., 2004; López‐Bendito et al., 2004; Ma, Hu, Berrebi, Mathers, & Agmon, 2006; Oliva, Jiang, Lam, Smith, & Swann, 2000; Silberberg et al., 2016; Tamamaki et al., 2003; Wang et al., 2009; Zhang, Kaneko, Yanagawa, & Saito, 2014). Recently developed TgN(GAD65‐tdTomato) mice show robust expression of the RFP tdTomato in GABAergic neurons, but tdTomato is expressed at lower levels in the cerebellum of TgN(GAD65‐tdTomato) and its expression was undetectable in glycinergic neurons (Besser et al., 2015). Moreover, because the most widely used tools in neuroscience utilize green fluorescence (e.g., Fluo4, Oregon Green BAPTA, and channelrhodopsin‐YFP), the application of these tools to cerebellar inhibitory neurons and glycinergic neurons remains difficult. Furthermore, these Tg mouse lines have the following potential limitations: (a) difficulty in discriminating homozygous/heterozygous animals and (b) unexpected adverse effect(s) caused by transgene‐induced genomic modifications. The identification of the locus in which the transgene has inserted could circumvent these limitations and provide tools to monitor the epigenetic condition of the inserted locus. Thus, there is still strong demand for a novel mouse line that expresses RFP in inhibitory neurons and has a known transgene insertion locus.
Another limitation is that most Tg/KI mouse lines express fluorescent proteins in the vast majority of the defined subsets of neurons. However, sparse fluorescent labeling of neurons enables the visualization of the fine structures of axons and dendrites using light microscopy. One remarkable example is the Thy1‐GFP/YFP Tg mouse lines (Feng et al., 2000), which enable the visualization of the spine and axonal morphologies of a defined subset of excitatory neurons.
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