The effect of acute or repeated stress on the corticotropin releasing factor system in the CRH-IRES-Cre mouse: A validation study
Introduction
Corticotropin releasing factor (CRF) is a key brain mediator of stress-related behaviors. A disruption in homeostasis of the CRF system has been linked to a variety of stress-related psychiatric disorders including depression, anxiety and addiction, all of which involve altered emotional states (Nemeroff et al., 1984, Henckens et al., 2016, Koob and Volkow, 2016). Manipulation of the CRF gene promoter has been accomplished using a variety of technologies, including bacterial artificial chromosome (BAC) transgenics (Alon et al., 2009) and genetic targeting (e.g. knock-in) (Kono et al., 2017), such as IRES-Cre (Taniguchi et al., 2011). The Crh-IRES-Cre mouse has been bred with several reporter lines including Ai3 (GFP) Ai9 and Ai14 (tdTomato), to generate mice with visualizable CRF-expressing cells (Wamsteeker Cusulin et al., 2013, Chen et al., 2015, Hooper and Maguire, 2016, Martin et al., 2010, Peng et al., 2017, Dedic et al., 2018). These neuronal populations are easily available for anatomical and morphological examination as well as electrophysiology and/or optogenetic/chemogenetic manipulation.
A major obstacle in identifying CRF neurons has historically been due to the difficulty in CRF peptide detection by immunohistochemistry using anti-CRF antibodies without additional tools, such as colchicine pre-treatment (Gunn et al., 2013, Borisy and Taylor, 1967). Colchicine is toxic at typically employed doses, leading to physical stress and neuronal activation in stress-related brain regions (Ceccatelli et al., 1989). The recent development of the Crh-IRES-Cre mouse has allowed for examination and manipulation of the CRF system and is emerging as a useful tool to examine the role of the mouse CRF system in stress-associated behaviors (Wamsteeker Cusulin et al., 2013, Dedic et al., 2018, Füzesi et al., 2016, Kondoh et al., 2016, Partridge et al., 2016). Recent studies have examined the validity of reporter expression in several key brain regions of the Crh-IRES-Cre::Ai14 and Crh-IRES-Cre::Ai9 mouse, including the bed nucleus of the stria terminalis (BNST), paraventricular nucleus of the hypothalamus (PVN), hippocampus and central nucleus of the amygdala (CeA) (Wamsteeker Cusulin et al., 2013, Chen et al., 2015, Dedic et al., 2018). Additionally, a recent study has mapped global reporter expression in the Crh-IRES-Cre::Ai3 mouse (Peng et al., 2017). However, some studies suggest that mouse lines crossed to different reporter lines show reporter expression in distinct populations or subpopulations within specific brain regions (Hooper and Maguire, 2016). For example, crossing of the BAC CRF-Cre line to either a tdTomato or mTomato-GFP reporter line led to labelling of anatomically distinct neuronal populations within the hippocampus (Hooper and Maguire, 2016). Therefore, as the Crh-IRES-Cre::Ai14 mouse has emerged as a popular mouse line to assess and manipulate the CRF system, further mapping and validation of expression is required. Thus, we mapped the distribution of tdTomato reporter in the Crh-IRES-Cre::Ai14 mouse and assessed the concordance of CRF protein and reporter expression throughout select brain regions.
Key to the role of CRF in stress responsivity is the hypothalamic–pituitary–adrenal (HPA) axis (Herman and Cullinan, 1997). CRF is released into the periphery from the PVN and directly activates the HPA axis by triggering the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary, which in turn stimulates the synthesis and secretion of glucocorticoids from the adrenal gland (Vale et al., 1981). Central administration of CRF induces anxiety-like behavior in the conflict, social interaction, acoustic startle and elevated plus maze tests (Swerdlow et al., 1986, Dunn and File, 1987, File et al., 1988). Further, both physical and psychological stressors activate CRF PVN neurons (Dayas et al., 2001). CRF is also located outside the PVN to control autonomic and behavioral responses to stress. For example, CRF-like-immunoreactivity is present in the neocortex, medial prefrontal cortex (mPFC), BNST, amygdala, medial septum, hypothalamus, thalamus, cerebellum, midbrain and hindbrain nuclei (Swanson et al., 1983). Additionally, the amygdala is thought to play a primary role in mediating anxiety-like responses (Davis et al., 2003). Regulatory dysfunction in the amygdala can lead to an abnormal pattern of affective responses and the development of recurring emotional disorders such as anxiety, depression and addiction (Gilpin et al., 2015).
Previous research has shown that distinct stressors can differentially modulate CRF expression and neural activation (See McReynolds et al., 2018 for review). For example, chronic unpredictable stress, but not acute restraint stress, increases CRF-like-immunoreactivity within the periventricular nucleus and anterior hypothalamic nucleus (Chappell et al., 1986). Additionally, acute, but not chronic, stress decreases CRF-like-immunoreactivity within the medial preoptic area (MPO) (Chappell et al., 1986). The specific pattern of activation of CRF neurons following acute and repeated psychological and pharmacological stress in mouse models remains uncharacterized. Here we examined the activation of CRF neurons in hypothalamic and extrahypothalamic brain regions following exposure to acute psychological (forced swim) and pharmacological (yohimbine) stressors in mice with and without a history of repeated restraint stress.
Section snippets
Animals
All studies were performed in accordance with the Prevention of Cruelty to Animals Act (2004), under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia (2013) and approved by The Florey Animal Ethics Committee. B6(Cg)-Crhtm1(cre)Zjh/J (Crh-IRES-Cre) mice and B6.Cg-Gt(ROSA)26Sortm14(CAG−TdTomato)Hze/J (Ai14) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; stock numbers
Crh-IRES-Cre::Ai14 distribution map
The distribution of tdTomato-expressing CRF cells was examined throughout the entire brain of male and female Crh-IRES-Cre::Ai14 mice. The density of tdTomato expression (per mm2) and % tdTomato-positive DAPI cells were quantified in 65 brain regions (summarized in Fig. 1 and Fig. 2). Two-way ANOVA showed a main effect of region for both tdTomato density (F(64,224) = 106.4, p < 0.001) and % tdTomato-positive DAPI cells (F(64,224) = 44.38, p < 0.001), but no effect of sex on density (F(1,224)
Discussion
CRF has a well-known role in stress responsivity, however, it is also involved in normal and pathological cognitive and emotional states including learning and memory (Nemeroff et al., 1984, Heim et al., 2009, Koob and Volkow, 2016). In this study, we examined the distribution of tdTomato-expressing CRF cells throughout the Crh-IRES-Cre::Ai14 mouse brain and reporter concordance with CRF peptide expression in selected brain regions. Further, we utilized the Crh-IRES-Cre::Ai14 mouse to examine
Funding and disclosure
All authors report no conflict of interest.
This work was supported by a National Health and Medical Research Council Project Grant to AJL (1079893). AJL is a National Health and Medical Research Council Principal Fellow (1116930). LCW is supported by the Australian Government Research Training Program Scholarship. EJC is supported by the University of Melbourne Early Career Researcher Grant Scheme. We acknowledge the Victorian State Government Operational Infrastructure Scheme.
Author contributions
LCW, EJC and AJL designed the study; LCW, EJC, and LC conducted all experiments; LCW and EJC conducted analyses; and LCW, EJC and AJL wrote the manuscript. All authors reviewed the content and approved the final version of the manuscript.
Data accessibility
For additional information on data analysis or generation, please contact the corresponding author.
Acknowledgements
We thank Dr. Sarah Ch'ng and Dr. Craig Smith for their technical assistance; Dr Wylie W. Vale (in memoriam), Dr Paul E. Sawchenko and Dr Joan Vaughan from the Salk Institute for Biological Studies for their generous gift of the antisera against CRF (PBL#C70).
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