Research reportExpression of channelrhodopsin-2 localized within the deep CA1 hippocampal sublayer in the Thy1 line 18 mouse
Introduction
Optogenetic methods have enabled significant advances in neuroscience, particularly in understanding complex circuit interactions (Zhang et al., 2010, Zhao et al., 2011, Kohara et al., 2014), and behavioral functions (Jasnow et al., 2013, Ting and Feng, 2013, Allen et al., 2015). The generation of transgenic animals for optogenetics has provided stable lines with reliable expression patterns between animals (Zhang et al., 2010, Fenno et al., 2011, Ting and Feng, 2013). Further, increased control over the expression of opsins can limit expression to precise neuronal populations, or cell types, through the use of highly specific promoters (Zhang et al., 2010, Fenno et al., 2011, Zeng and Madisen, 2012, Asrican et al., 2013, Ting and Feng, 2013).
Thy1 was the first of such promoters used to drive expression in transgenic optogenetic animals (Arenkiel et al., 2007), and is still broadly used today (Zhao et al., 2008, Porrero et al., 2010, Fenno et al., 2011, Chen et al., 2012, Asrican et al., 2013, Ting and Feng, 2013). There are a variety of Thy1 transgene founder lines with lines 9 and 18 being the most commonly used (Ting and Feng, 2013). These lines exhibit ChR2 expression in a variety of brain regions, including amygdala, hippocampus, and cortex (Fenno et al., 2011, Asrican et al., 2013, Ting and Feng, 2013). While expression patterns of transgenes are specific and consistent within lines, expression patterns between lines differ. The distribution of Thy 1 transgenes between founder lines has been described in detail by Feng et al. (2000), and others (Arenkiel et al., 2007, Asrican et al., 2013, Ting and Feng, 2013). Transcription of the Thy1 gene dramatically decreases postnatally, contributing to slight variation in expression. This increased variance in expression patterns of Thy1 transgenes among brain regions has been attributed to differential induction of silencing mechanisms, dependent on copy number of transgenes and state of chromatin condensation proximal to their insertion point (Feng et al., 2000).
Thy1-ChR2 line 18, with high expression levels in area CA1, is a particularly useful model for studying hippocampal physiology. Gross histological examination suggests that ChR2 is primarily expressed in CA1 pyramidal neurons (Arenkiel et al., 2007, Asrican et al., 2013). However, there are relatively few studies which have examined cell-type specific expression, and reports in some Thy-1 founder lines indicate expression of ChR2 may be present in interneurons of CA1 (Asrican et al., 2013, Ladas et al., 2015). There are more than 21 subtypes of interneurons in CA1, each playing different functional roles in hippocampal microcircuits (Freund and Buzsaki, 1996, Klausberger and Somogyi, 2008, Bezaire and Soltesz, 2013). In addition, the pyramidal cell population is not a single homogenous cell layer, but rather composed of at least two sublayers that are differentiable based upon physiological features and expression of the calcium buffering protein calbindin (CBN) (Mizuseki et al., 2011, Kohara et al., 2014, Valero et al., 2015). These sublayers receive distinct inputs and are thought to represent different functional pathways within CA1. To identify the specific expression of ChR2 in the hippocampus of the Thy1, line 18 mouse we used cell-type specific markers to perform a histological examination of select interneurons and pyramidal cell sublayers. Our results demonstrate that expression in CA1 is specific to CBN negative, deep pyramidal cells. These findings highlight the utility of Thy1-ChR2-YFP, line 18, mice to generate a better understanding of the role of this specific subset of neurons in hippocampal circuitry.
Section snippets
Animals
All animals were maintained at Wake Forest University School of Medicine on a 12 h light/dark cycle. All experiments were approved by the Wake Forest University Animal Care and Use Committee and complied with National Institute of Health guidelines for minimizing pain and discomfort. Thy1-ChR2-YFP, line 18, mice were obtained from Jackson Laboratory (B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J, stock number 007612). Five adult mice, ranging from 8 to 12 months, were sacrificed using Euthasol (Virbac, Fort
Results
Expression patterns of Thy1-ChR2 and other Thy1 transgenes have been observed to differ between founder lines. Thy1 transgenes have been found to express in both excitatory and inhibitory cell types, or in certain lines, both cell types (Feng et al., 2000, Allen et al., 2015, Ladas et al., 2015). Thy1-ChR2-YFP line 18 has prominent expression in hippocampus, cortex, and amygdala (Arenkiel et al., 2007, Asrican et al., 2013, Ladas et al., 2015). For a more detailed examination of ChR2 expression
Discussion
Owing to the precise temporal and spatial regulation made possible by selective expression of opsins, optogenetics affords the ability to study a variety of functions with precise stimulation of neurons. However, different methods for inducing opsin channel expression can generate expression patterns with varying specificity. Our study illustrates the high degree of specificity in expression of ChR2 in the line 18 transgenic Thy1-ChR2-YFP mouse, at least within CA1 of the hippocampus.
Within
Conflict of interest
Authors declare no conflict of interest for the work presented in this manuscript.
Funding
This study is supported by NIAAA R01AA016852 and NIAAA T32AA007565.
Acknowledgments
The authors are grateful for the use of the Wake Forest Biology Microscopic Imaging Core Facility. We would also like to thank Dr. Glen Marrs for his assistance and training with image acquisition and analysis and Greg Alberto for his careful review of this manuscript.
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