The glucocorticoid receptor (GR) is a ligand (glucocorticoid)-activated transcription factor that participates widely in functions related to homeostasis and adaptation (Herman, 1993). Functional properties of GR action have been widely queried, using pharmacological, viral vector and electrophysiological approaches, with most of the literature using rat models (McKlveen et al., 2013; McKlveen et al., 2016; Ghosal et al., 2014; Solomon et al., 2014; Wulsin et al., 2010). Recent decades have seen an increase in the use of mouse models to provide Cre-driver mediated deletion in specified cell populations (e.g. CaMKIIα neurons of the forebrain, Simpleminde-1 (Sim-1) neurons of the hypothalamus; Nahar et al., 2015; Solomon et al., 2015). While yielding valuable information on cell type-specific GR actions (Solomon et al., 2012), the use of mice has proven problematic with regard to high-resolution behavioral and physiological analyses due to the different behavioral repertoire of mice (high strain and inter-experiment variability in memory tests) and the small size (e.g., prohibits blood sampling of large volumes) (Whishaw and Tomie, 1996; Parker et al., 2014; Ellenbroek and Youn, 2016; Colacicco et al., 2002).

The emergence of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas 9-mediated gene editing now affords gene targeting in numerous species, including rat (Wang et al., 2016; Shao et al., 2014; Ran et al., 2013). For example, CRISPR/Cas9 methods allow for introduction of exon-flanking LoxP sites to generate conditional knockdown alleles and subsequent gene deletion following exposure to Cre recombinase using either driver lines or viral vector approaches. In this study, we used CRISPR/cas9 to specifically insert LoxP sites to sequences flanking exon 3 of the Nr3c1 (GR) gene in Sprague Dawley rats (an outbred strain commonly used to explore GR function) via homology-directed repair (HDR). Our data provide validation of the Nr3c1 gene editing in rats, and the use of viral vector-mediated Cre delivery to demonstrate targeting of GR knockdown to specific cell types, brain regions and circuits. Functional efficacy of GR knockdown in the prelimbic division (PL) of the medial prefrontal cortex (PL-PFC) was verified by sex-specific deficits in extinction of conditioned fear in females and a shift to active coping in the forced swim test (FST) in males. Overall, our study shows the utility of this gene editing technique to generate conditional gene deletion models that can leverage the considerable advantage of rats in behavioral and physiological research.

We directly manipulated the genome of Sprague Dawley rat zygotes by CRISPR/Cas9 to generate the floxed Nr3c1 allele. We used a dual sgRNA strategy to delete the sequence containing exon 3 of the Nr3c1 gene and repaired it with a cutting resistant donor plasmid that contains the deleted sequence, two flanking LoxP sites at the cut location of the sgRNA recognition sites, a right homologous arm at 2.57 kb, and a left homologous arm at 1.95 kb (Figure 1A and B). Because truncated sgRNAs increase targeting specificity (Fu et al., 2014), we chose six sgRNAs with various lengths (17–20 nt) (Appendix 1—table 1) and validated their editing activity in rat C6 glioma cells by T7E1 assay (Appendix 1—figure 1). We picked two sgRNAs, sg-2 and sg-6, for targeting the 5’ and 3’ sequences of exon 3, respectively. Both sgRNAs were 17nt in length, which is expected to provide high specificity (Fu et al., 2014). The two selected sgRNAs, Cas9 mRNA, and the donor plasmid were microinjected into ~60 rat zygotes, followed by embryo transfer into pseudopregnant female rats. Seventeen pups were born. We identified that one of them (No. #60) was correctly targeted, which was confirmed by PCR with the external primers (Figure 1C and D) paired with the primers partially containing LoxP sequences (P5-P6 and P10-P8 for 5’ and 3’ ends, respectively; Figure 1E and F). It was further confirmed by primer pairs P5-P7 and P9-P8, followed by ClaI and BamHI enzyme digestion and Sanger sequencing (data not shown), and by additional Sanger sequencing of the entire targeted area between P5 and P8. This allele contains an A/T base change at chr18:31,744,353, compared to the rn6 reference genome, which is located in a non-conserved intronic region and unlikely to be of functional consequence. We verified copy number and used plasmid backbone PCR to exclude the possibility of random integration of the donor plasmid (Appendix 1—figure 2). We have consistently bred the offspring of rat #60 to homozygosity (Figure 1G).

Figure 1

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Validation of conditional GR knockdown: Viral vector targeting

To test the efficacy of this novel rat line, we administered adenoviral Cre recombinase constructs to drive regional, cell type-specific and projection-specific knockdown of GR. Regional knockdown targeted the basolateral amygdala (BLA), using human synapsin promoter-driven Cre recombinase (AAV8-hSyn-Cre, UNC Vector Core, NC, USA) microinjections. Cre⁺ cells were devoid of nuclear GR immunoreactivity in SD:nr3c1^fl/fl rats (Figure 2A), whereas the vast majority of Cre⁺ cells co-expressed GR in wildtype controls (SD:nr3c1^wt) (Figure 2B) injected with the same viral construct (AAV8-hSyn-Cre, UNC Vector Core).

Figure 2

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We then assessed cell-type specific knockdown by injection of AAV9.CamKII.HI.eGFP-Cre.WPRE.SV40 (CaMKIIα Cre) virus into the PFC of SD:nr3c1^wt or SD:nr3c1^fl/fl rats. Infusions of CaMKIIα Cre caused widespread loss of GR immunoreactivity within GFP⁺cells in the PFC of CaMKIIα Cre virus injected SD:nr3c1^fl/fl rats (Figure 2C yellow arrows), but no GR loss is noted in wildtype control (SD:nr3c1^wt) injected rats (Figure 2D white arrows) or in uninfected CaMKIIα cells in SD:nr3c1^fl/fl rats (Figure 2C white arrows).

Finally, we used an intersectional approach to examine connectional knockdown of GR, focusing on PFC projection neurons to the BLA. Retrogradely-infected (Cre⁺) neurons were observed in the PFC after administration of AAVrg pmSyn1-EBFP-Cre, (Addgene) to the BLA. We did not observe GR immunoreactivity in SD:nr3c1^fl/fl rats (Figure 2E), whereas substantial proportions of PFC-BLA projecting neurons contained GR immunoreactivity in control virus (AAVrg-CAG-GFP, Addgene) injected animals (Figure 2F). Although we made every effort to employ appropriate viral controls, we were limited by the availability of viral constructs, specifically AAV retrograde constructs, and this is a caveat of the current study. For our intersectional approach we used the same AAV serotype (AAVrg) for both knockdown and controls however, the promoter (pmSyn1 vs CAG) and fluorescent reporter (EBFP vs GFP) were different. These constructs were chosen as the best available at the time of running the experiments. These studies highlight the novel use of this rat model to query not only the role of GR in a specific region in isolation but also how GR functions as part of an integrated circuit.

Behavioral consequences of targeted GR knockdown; Implications for fear and coping behaviors

We next performed a functional test of viral Cre-mediated GR knockdown, focusing on the PL-PFC. SD:nr3c1^fl/fl (GRKD) rats and wild type littermate controls SD:nr3c1^wt (Control) all received injection of AAV9.CamKII.HI.eGFP-Cre.WPRE.SV40 (CaMKIIα Cre) [Penn Vector Core] into the PL-PFC Figure 3E. CaMKIIα is a calcium binding protein that is most commonly found in glutamatergic neurons of the forebrain (although there is evidence of the presence of CaMKIIα in some GABAergic interneurons in the amygdala and striatum and possibly the PFC; Jennings et al., 2013; Klug et al., 2012; Robison et al., 2014), thus we are targeting a glutamatergic enriched populations of neurons in the PL-PFC.

Figure 3

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We first confirmed GR knockdown in virus infected neurons in both females and males using immunohistochemistry [Females = T(13)=9.752; p<0.0001 (Figure 3A)]; [Males = T(13)=6.202; p<0.0001 (Figure 3B)].

The PFC plays a critical role in extinction of emotional memory (e.g. conditioned fear), selection of emotional coping strategy, and hypothalamic pituitary adrenal (HPA) axis reactivity (Amat et al., 2005; Giustino and Maren, 2015; Quirk et al., 2000; Quirk et al., 2006). We therefore tested whether GR knockdown in CaMKIIα targeted neurons in this region affected extinction of conditioned fear to an auditory fear conditioning paradigm, and behavioral coping during the FST. For fear conditioning, rats were exposed to five tone shock pairings on the first day (acquisition), followed by 2 days of 20 tones without a paired shock, (extinction and extinction recall). Data was binned for clarity into five tones per bin. Freezing during the tones was measured as fear behavior. Freezing in female rats with PL-PFC targeted GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) increased in acquisition [GRKD F(1,52) = 5.553; p=0.035], but there was no significant interaction effect [GRKD x tone F(4,52) = 1.292; p=0.285] (Figure 4A). Extinction of fear conditioning was delayed in the female GRKD group relative to controls, as indicated by a significant time x GRKD interaction effect [GRKD x time F(3,39) = 4.184; p=0.012] (Figure 4A). Extinction recall was also impaired in female PL-PFC GRKD rats relative to controls (significant time x GRKD interaction) [GRKD x time F (3,31)=3.796; p=0.020] (Figure 4A). Meanwhile, males with PL-PFC targeted GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) did not differ from controls [Acquisition GRKD x tone F(1,79]=1.827; p=0.136] [Extinction GRKD x tone F(1,63) = 0.873; p=0.463] [Extinction Recall GRKD x tone F(1,63) = 0.592; p=0.624] (Figure 4B). While most lesion and inactivation studies have shown that the PL-PFC is critical for appropriate fear responding (Giustino and Maren, 2015; Quirk et al., 2000; Quirk et al., 2006), the use of the GR floxed rat model has shown that GR in the CaMKIIα neurons of the PL-PFC may be more critical for female expression and extinction of conditioned fear, and less so for males.

Figure 4

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The PFC has also been implicated in coping behaviors and behavioral adaptation during acute and chronic stress. We used the FST to investigate remodeling of behavioral coping strategy during an acute stress challenge. There was no effect of PL-PFC-driven GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) on immobility time in the FST on either day 1 or day 2 for male rats [Day 1 immobility T(14) = −1.8.48; p=0.085] (Figure 4C) [Day 2 immobility T(14) = −1.411; p=0.180] (Figure 4D). However, males with CaMKIIα-driven PL-PFC GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) increased diving frequency on day 1 [T(14)=3.252; p=0.005] (Figure 4E) relative to controls, consistent with an altered coping strategy. This behavior persisted into the second day of the FST test, with significantly higher number of dives for males with PL-PFC GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) [T(14) = - 3.468; p=0.003] (Figure 4F). Females with PL-PFC GRKD (SD:nr3c1^fl/fl plus CaMKIIα Cre) did not differ from controls in total immobility (Figure 4G–H) or number of dives (Figure 4I–J) [Day 1 Immobility, T(13) = 0.077; p=0.939] [Day 2 immobility, T(13) = −1.083; p=0.298] [Day 1 dives, T(13) = −0.187; p=0.854] [Day 2 dives, T(13) = −1.316; p=0.211]. Although there were no changes in total immobility in the FST, increased diving in the males with PL-PFC GRKD suggests GR in CaMKIIα neurons of the PL-PFC may facilitate a shift, selectively in males, to more diverse active escape behaviors than just swimming and climbing alone.

Our lab has previously shown that GRKD in the PL-PFC increases corticosterone to acute restraint (McKlveen et al., 2013). We subjected male and female GRKD and control rats to a 30min acute restraint challenge and took tail blood samples at 0 min, 15 min, 30 min, 60 min, and 120 min from the start of restraint to measure plasma corticosterone. There was a GRKD x time interaction [F(4,45)=3.020; p=0.027] in females, with post hoc analysis revealing delayed shut-off (120 min time point) of the HPA axis stress response in PL-PFC-GRKD rats [p=0.050] (Figure 4K). There was no effect of GRKD [F(1,44)=2.046; p=0.180] or GRKD x time interaction observed in males [F(4,44)=0.935; p=0.452] (Figure 4L). The data suggest that female SD:nr3c1^fl/fl rats with PL-PFC GRKD may have deficits in negative feedback inhibition due to the inability to return to a similar resting level of corticosterone as control rats. The lack of differences after PL-PFC GRKD in the males compared to our previous study may be due to the specificity of our virus to knockdown primarily in CaMKIIα cells, whereas previously a ubiquitous promotor on a lentiviral delivered shRNA was used which targets all neuronal cell types, including inhibitory GABAergic interneurons (McKlveen et al., 2013).

Prior studies document generation of knockdown and Cre dependent knockdown rat models using CRISPR/Cas9 as a molecular tool (Shao et al., 2014; Ma et al., 2014) reviewed in Wang et al. (2016). Here we created a conditional (Cre-recombinase dependent) GR knockdown rat using CRISPR/Cas9. Validation of genome integration was accomplished by PCR, and Cre-dependent knockdown by site and circuit specific viral vector expression of Cre-recombinase. Finally, efficacy and consequences of GR knockdown in the PL-PFC were supported by sex-specific effects in behavior. The CaMKIIα promoter is thought to largely direct Cre expression to cortical projection neurons with over 80% specificity and efficacy (Wood et al., 2019) and thus it is likely our manipulations were able to successfully target a cell population composed mostly of excitatory projection neurons.

Deficits in fear conditioning and alterations in behavioral coping style are a common phenotype in murine GR knockdown models as well as following chronic stress (McKlveen et al., 2013). We used site and cell specific knockdown of GR to demonstrate that CaMKIIα cells in the PL-PFC require GR for appropriate fear responses and extinction in females. Conversely, male rats lacking GR in the PL-PFC, shift to an active escape behavior in the FST, suggesting adoption of an active coping strategy. Females (but not males) evidenced deficient shut-off of the HPA axis stress response, consistent with a sex-specific dependence of PL-PFC GR for full feedback inhibition. The data highlight a strong interaction between GR signaling and sex in coordination of prefrontal cortical signaling mechanisms. Moreover, the data provide functional evidence that CRISPR/Cas9 can be used to provide high-resolution cell and site specific assessment of GR action in brain, leveraging the advantages of the rat as a model organism.

Prior studies have used promoter-specific Cre driver mouse lines to generate targeted GR knockdown in mice. Mouse studies demonstrated that CaMKIIα-Cre directed GR deletion in the forebrain (targeting exon 2 or 3) promoted anxiety-related behavior, passive coping, and corticosterone hypersecretion in male, but not female mice (Solomon et al., 2012; Boyle et al., 2006; Hartmann et al., 2017). It is important to note that the mouse CaMKIIα driver line deletes GR in multiple brain regions, including cortex, hippocampus, BLA, caudate, and bed nucleus of the stria terminalis, many of which interface with stress and emotional behavior. The extensive knockdown makes it impossible to specify circuit-specific roles of stress hormone signaling in behavior and stress physiology. Prior studies have not specified GR-specific deficits in cognitive behaviors, which can be difficult to assess in mouse models (Whishaw and Tomie, 1996; Parker et al., 2014; Ellenbroek and Youn, 2016). Here, we show that by using these viral constructs with specific promoters (CaMKIIα) in GR floxed rats, we can not only drive expression of Cre-recombinase in a cell-type specific manner, but also a defined region, allowing precise investigation of the role of GR in behavior.

There is a possibility of genetic modifications, even with the highly site specific editing of the CRISPR/Cas9 system, when inserting LoxP sites into the genome (reviewed in Zhang et al., 2015; Fu et al., 2013; Cho et al., 2014). We tested the most likely frameshift insertion and deletion (indel) mutations that could be introduced by unexpected non-homologous end joining or inaccuracies in the sgRNA targeting. We used PCR amplification and sequencing to determine that there were no mismatches in these sequences compared to wildtype controls (Appendix 1—table 2 and Supplementary file 1). We are thus confident that our LoxP insertion did not cause any genetic mutations that would interfere with in vivo studying of the GR functioning in the rats on a genetic basis. As an additional control, we further investigated physiological and behavioral effects of this targeted insertion. We performed multiple tests of behavior (open field, elevated plus maze, and FST), and observed no effect of gene targeting on any behavioral endpoint (Appendix 1—figure 3). Furthermore, we found no differences in bodyweight (Appendix 1—figure 4) or organ weights commonly changed with chronic stress (Appendix 1—table 3).

Our functional studies indicate that female (but not male) rats with knockdown of GR in the PL-PFC have heightened fear response and impaired extinction. Importantly, these data inform prior PL-PFC lesion/inactivation studies, which indicate that the PL-PFC is critical for appropriate encoding and expression of conditioned fear (Quirk et al., 2000; Morgan and LeDoux, 1995; Marquis et al., 2007). The data indicate that GR signaling is an important component of this process in females, but may be less so in males (although the increased freeze times in males may reflect a more ‘intense’ response to the shock, which may result in ceiling effects on subsequent exposure to cues). In the FST, males but not females increased the number of diving events. As diving is considered an active coping behavior (Bielajew et al., 2003; Fujisaki et al., 2003; Molina et al., 1994), it is likely that GR in the PL-PFC also interfaces with coping behaviors in a sex-specific manner. In combination, these data indicate that use of enhanced precision methods of gene targeting such as CRISPR/Cas9 in rat reveal substantive new information on the biology of the PL-PFC and its interaction with biological sex.

Deficits in conditioned fear and glucocorticoid feedback efficacy are associated with depression, post-traumatic stress disorder and other stress related diseases in human. It is important to consider that these disease states are overrepresented in women (McKlveen et al., 2013; Herman, 1993), and also involve modification of prefrontal cortical circuitry in neuroimaging studies (Breslau et al., 1997; Breslau and Anthony, 2007; Kessler et al., 1995; McLean et al., 2011). Emergence of behavioral deficits following targeting of GR in the PL-PFC of females may reflect a mechanism underlying selective vulnerability of females to stressful life events, suggesting that appropriate GR signaling is required to mitigate the impact of adversity.

Overall, we demonstrate that CRISPR/Cas9 gene editing is effective in generating novel tools for bio-behavioral research in a highly tractable model organism with a long and well-documented history. As noted in our functional studies, the SD:nr3c1^fl/fl line can be of significant value in the context of higher order behavioral assays (cognitive behaviors, goal-directed behaviors, reward behaviors/drug self-administration) that can be often difficult to implement and/or interpret in other rodent organisms, such as mice. With the growing number of viral Cre constructs and the recent surge in development of rat Cre driver lines, the CRISPR/Cas9 method can provide site and cell type gene deletions or manipulations that are vital for our understanding of mechanisms of stress pathologies and stress-related diseases.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Jessie R Scheimann

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Rachel D Moloney

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7912-4898

2. Rachel D Moloney

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Jessie R Scheimann

   For correspondence

   Rachel.Moloney@uc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7111-3414

3. Parinaz Mahbod

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Rachel L Morano

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Maureen Fitzgerald

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Olivia Hoskins

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Benjamin A Packard

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Conceptualization, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. Evelin M Cotella

   Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

   Contribution

   Software, Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

9. Yueh-Chiang Hu

   1. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
   2. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, United States
   3. Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, Cincinnati, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

10. James P Herman

    Department Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, United States

    Contribution

    Conceptualization, Resources, Software, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    James.Herman@uc.edu

    Competing interests

    No competing interests declared

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