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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Thank you for submitting your article "Use of CRISPR/Cas9 gene targeting to conditionally delete glucocorticoid receptors in rat brain" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Geoffrey Schoenbaum as the Reviewing Editor and Catherine Dulac as the Senior Editor. The following individuals involved in review of your submission have also agreed to reveal their identity: Brandon K Harvey (Reviewer #1) and Carmen Sandi (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In the current study, the authors report the development of a conditional deletion of the GR in rats. They provide credible evidence that their tool works, affects GR conditionally and that this impacts behavior as expected. The reviewers agreed that having such a tool available in rats would be a substantial step forward for the field. There are concerns but they are requests for details or changes in emphasis, none of which should take more than a few months. If the paper is revised with the additional details, the reviewers agreed that it would be acceptable for publication.

Essential revisions:

The essential revisions are targeted at providing additional detail that was thought to be critical to the validity of the tool. Other details requested in the reviews are important, but these are particularly critical as they support the rationale for publishing. There are three:

1) Correcting the emphasis on CRISPR as raised by reviewer 1.

2) Immunostaining patterns comparing effects in males and females as raised by reviewer 2.

3) Sequencing of PCR amplicons for the entire integrated transgene as raised by reviewer 1.

Reviewer #1:

The authors have developed a conditional knockout rat based on the Cre-loxP system to remove exon 3 of the Nr3c1 gene encoding the glucocorticoid receptor. They employ CRISPR/Cas9 in the development of a conditional knockout rat for the Nr3c1 gene. There is an overemphasis on the use of CRISPR/Cas9 starting with the title which is misleading in its current form. The conditional deletion of GR in rat brain is conditional on Cre not Cas9. Additionally, there is no evidence that CRISPR was required to make the animal. They obtained 1 animal out of 60 with the transgene. This could occur by injecting the targeting plasmid (as was done prior to the now common use of CRISPR for increased targeting). It is likely CRISPR aided in the process but there is no evidence for this (e.g. a plasmid alone was not injected and for good reason). The authors also do no appear to have done any PCR/Sequence/T7 assay/TIDE analysis on any of the 59 negative animals for evidence of CRISPR-induced mutation (which may have provided some possibly interesting GR mutants but this was not the goal). CRISPR should be deemphasized and focus on the fact there is a new Cre-dependent KO for GR and some possibly interesting biological observations using this new resource.

The authors claim in the first paragraph of the Results section that offspring fecundity is evidence of no random integration. This is not the case. There may be random integration that doesn't affect breeding but disrupts other gene function. A copy number per genome determination is needed. A PCR assay internal to the transgene that uses one of the primers partially overlapping the loxP site to avoid amplification of endogenous locus and a PCR assay to a single copy gene in the genome (2 copies per genome) should be used for QPCR to determine transgene copy number per genome. This assay is becoming a standard practice in transgenic animal development. Also, if the entire plasmid was injected, PCR assays designed to detect other plasmid segments is recommended.

The authors do not appear to have sequenced PCR amplicons for the entire integrated transgene. This is important to show no mutations or aberrant repair occurred at the single nucleotide level. Years of work may be built on these animals that have a point mutation in exon 3 altering function of GR. Alignment of sequencing data to in silico predicted sequence should be provided as supplement.

The use pf AAVrg-CAG-GFP as control for AAVrg pmSyn1-EBFP-Cre is not clear. These are different promoters and transgenes (e.g. using a "dead" Cre in the same construct would be preferred). Consistent with other experiments, AAVrg pmSyn1-EBFP-Cre should be injected into WT animals as control.

Overall, the finding of GR knockout in the prelimbic area affecting behavior is mostly confirmatory with published work while providing some interesting phenomenological observations for future studies. As such, this is a methods paper describing a transgenic rat with much potential and may be better in a specialized journal or methods section of neuroscience journal.

Reviewer #2:

Scheimann et al. generated a conditional GR knockout in rats using CRISPR/Cas9 technology. They provide evidence of targeted GR deletion in specific cell types, brain regions, and circuits via viral vector-mediated delivery of Cre. They also report that deletion of GR in output neurons of the prelimbic cortex resulted in sex-specific deficits in extinction of conditioned fear in females and a shift to active coping in the forced swim test in males. The manuscript is well-written and development of a conditional GR knockout model in rat is important and will be useful tool for probing GR function in the brain. However, there are several major limitations to the data that should be addressed to strengthen the conclusions of the manuscript.

1) Given the sex-specific effects on behavior, it is critical to show and evaluate the immunostaining patterns from both male and female rats. Are sex-specific differences observed in the distribution, efficiency, and/or extent of GR knockout in male and female brains?

2) Does the loss of GR lead to compensatory increases in the expression of the related receptor MR? This is an important consideration given that MR binds corticosterone, is co-expressed with GR in many brain regions, and is known to play a role in stress and emotional behavior.

3) Several issues should be addressed concerning Figure 2:

a) Why is the immunostaining pattern for GR in panel C different than in panel A? For the GR knockout cells in panel C, it appears that GR immunostaining is lost in the nucleus but not the cytoplasm. Why is this the case?

b) The authors use AAVrg-CAG-GFP injection of SD:nr3c1fl/fl rats as a control for the AAVrg pmSyn1-EBFP-Cre injection (panels E and F). Another important control is injection of AAVrg pmSyn1-EBFP-Cre into wild-type rats. Was this control performed?

c) The images should be enlarged. The small size of the images makes it very difficult to evaluate the staining patterns.

4) Does knockout of GR in the prelimbic cortex alter locomotor activity of the rats?

5) The sample size for the GR knockout rats is quite small (n = 5 for females and n = 6 for males) for behavioral studies. In addition, a Fisher's LSD test was employed for post-hoc analyses. Are similar significance results found when statistical tests are utilized that correct for multiple comparisons?

Reviewer #3:

This is the first study that develops a rat model with the capacity to perform cell- and region-specific deletion of the glucocorticoid receptor (GR) in rats. In addition to the novel tool development, the authors provided evidence of the behavioral impact of specific deletion of GR in the rat prelimbic CamKII-(glutamatergic) containing neurons. The data indicates differential effects of the knockdown approach on fear conditioning and extinction and behavior in the forced swim test in males and females, with a minor effect on corticosterone recovery in females. The study seems to be well performed and it is clearly written. Rats as model organisms have proved superior to mice for the dissection of stress (neuro)physiology and behavior and the current resource is a major step forward for the interrogation of GR function in this animal species.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Conditional deletion of glucocorticoid receptors in Rat brain results in sex specific deficits in fear&coping behaviors" for further consideration at eLife. Your revised article has been favorably evaluated by Catherine Dulac (Senior Editor), a Reviewing Editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Please remove the statement concerning reviewer 1 below, and make certain the second issue raised is clearly noted as a caveat of the study if more data cannot be provided to address this directly.

Reviewer #1:

Below in quotes are the authors' responses to previous concerns. A follow up response is given. All other concerns were adequately addressed.

Authors: "…addition, we would like to clarify that we described the fact "the offspring of rat #60 can be bred to homozygosity indicates the correct targeting of loxP sequences to the Nr3c1 gene, instead of random integration". We did not mention anywhere in the text that offspring fecundity is evidence of no random integration."

My response: Although it is true the authors don't use the same verbiage, the ability to produce homozygous offspring should not be used as an indicator of correct targeting. The integration could still have additional integrants which would likely would be outcrossed if on a different chromosome or at least a sufficient distance from the targeted allele being genotyped. However, the only way to verify is to assay for such events and the authors have now done a more rigorous analysis to characterize the transgene integration. The statement in quotes provides an unnecessary precedent and contributes to the propagation of inadequate characterization of novel transgenic animals. It is not needed especially with the additional data and should be revised as simple statement of fact without indication or just remove it.

Authors: "The authors acknowledge that the use of these 2 viruses may not be entirely clear. The viruses used for these experiments were sourced from Addgene. At the time these experiments were performed, this combination of "control" and "Cre" viruses were the best available. The AAVrg pmSyn1-EBFP-Cre virus was not available in a non-Cre construct nor was the AAVrg-CAG-GFP virus available with a Cre insert. Addgene has now updated its inventory and the AAVrg pmSyn1-EBFP-Cre is no longer available and so we are unable to perform surgeries in WT animals at this time as suggested by the reviewers."

My response: Given that GFP expression has been demonstrated to cause toxicity, the concern is that any differences observed may be due to differences in viral transgene expression. Do AAVrg-CAG-GFP injected animals have same behavior as uninjected animals? At minimum, a strong emphasis must be placed on this caveat of the study. This is the only major concern remaining.

Reviewer #3:

The authors have addressed all my previous points satisfactorily. I do not have any further issues. The paper represents an important addition and tool to the field.

Essential revisions:

The essential revisions are targeted at providing additional detail that was thought to be critical to the validity of the tool. Other details requested in the reviews are important, but these are particularly critical as they support the rationale for publishing. There are three:

1) Correcting the emphasis on CRISPR as raised by reviewer 1.

2) Immunostaining patterns comparing effects in males and females as raised by reviewer 2.

3) Sequencing of PCR amplicons for the entire integrated transgene as raised by reviewer 1.

Reviewer #1:

The authors have developed a conditional knockout rat based on the Cre-loxP system to remove exon 3 of the Nr3c1 gene encoding the glucocorticoid receptor. They employ CRISPR/Cas9 in the development of a conditional knockout rat for the Nr3c1 gene. There is an overemphasis on the use of CRISPR/Cas9 starting with the title which is misleading in its current form. The conditional deletion of GR in rat brain is conditional on Cre not Cas9. Additionally, there is no evidence that CRISPR was required to make the animal. They obtained 1 animal out of 60 with the transgene. This could occur by injecting the targeting plasmid (as was done prior to the now common use of CRISPR for increased targeting). It is likely CRISPR aided in the process but there is no evidence for this (e.g. a plasmid alone was not injected and for good reason). The authors also do no appear to have done any PCR/Sequence/T7 assay/TIDE analysis on any of the 59 negative animals for evidence of CRISPR-induced mutation (which may have provided some possibly interesting GR mutants but this was not the goal). CRISPR should be deemphasized and focus on the fact there is a new Cre-dependent KO for GR and some possibly interesting biological observations using this new resource.

We appreciate the reviewer’s comments and have now changed the title to more accurately describe the content of the paper. We also wanted to clarify that in the first paragraph of the Results section, we described that we obtained 1 Nr3c1 floxed animal out of 17 pups, as a result of microinjection of 60 fertilized eggs. This Nr3c1 floxed animal was #60, which may have caused the viewer to think that 1 correctly targeted animal was obtained from 60. Therefore, we changed #60 to No. #60 in the text. In addition, the rest of 16 negative animals carried the alleles, including single loxP, large deletion between two sgRNA target sites, and indels at individual sgRNA target sites. As also mentioned by the reviewer, it was not our goal to study animals with a partially integrated transgene, indels at introns or knockout alleles (which is lethal) for the current study. Therefore, we do not show the genotyping data for the 16 negative animals in the manuscript.

The authors claim in the first paragraph of the Results section that offspring fecundity is evidence of no random integration. This is not the case. There may be random integration that doesn't affect breeding but disrupts other gene function. A copy number per genome determination is needed. A PCR assay internal to the transgene that uses one of the primers partially overlapping the loxP site to avoid amplification of endogenous locus and a PCR assay to a single copy gene in the genome (2 copies per genome) should be used for QPCR to determine transgene copy number per genome. This assay is becoming a standard practice in transgenic animal development. Also, if the entire plasmid was injected, PCR assays designed to detect other plasmid segments is recommended.

We fully agree with the reviewer’s comments and have presented the data showing the evidence of no random integration. We included two additional experiments in the revision. First, we performed a copy number analysis on three regions of the integrated area, including ‘5 to 5’ loxP, 3’ to 3’ loxP and between two loxP sites. We compared two WT and two homozygous Nrc31 floxed rats and confirmed that there are 2 copies per genome for each of the three regions examined (Appendix 1—figure 2). Secondly, because a circular DNA plasmid was used for microinjection to reduce the random integration, we performed the PCR using the primers encompassing the either end of the plasmid backbone sequence. Both reactions did not detect any signals (Appendix 1—figure 2), consistent with the copy analysis indicating the absence of random integration. In addition, we would like to clarify that we described the fact “the offspring of rat #60 can be bred to homozygosity indicates the correct targeting of loxP sequences to the Nr3c1 gene, instead of random integration”. We did not mention anywhere in the text that offspring fecundity is evidence of no random integration.

The authors do not appear to have sequenced PCR amplicons for the entire integrated transgene. This is important to show no mutations or aberrant repair occurred at the single nucleotide level. Years of work may be built on these animals that have a point mutation in exon 3 altering function of GR. Alignment of sequencing data to in silico predicted sequence should be provided as supplement.

We appreciate the reviewer’s suggestion. We have performed the long-range PCR using the primers encompassing the entire transgene region and sequenced from end to end. We confirmed that the DNA from two homozygous Nr3c1 floxed animals showed no mutation in and near the coding sequence. However, we detected 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 next to a SINE transposable element. Given that SD rats are an outbred strain and the location of the base change, we do not expect that this particular base difference will cause any significant effect. We have documented it in the revision (Results paragraph one).

The use pf AAVrg-CAG-GFP as control for AAVrg pmSyn1-EBFP-Cre is not clear. These are different promoters and transgenes (e.g. using a "dead" Cre in the same construct would be preferred). Consistent with other experiments, AAVrg pmSyn1-EBFP-Cre should be injected into WT animals as control.

The authors acknowledge that the use of these 2 viruses may not be entirely clear. The viruses used for these experiments were sourced from Addgene. At the time these experiments were performed, this combination of “control” and “Cre” viruses were the best available. The AAVrg pmSyn1-EBFP-Cre virus was not available in a non-Cre construct nor was the AAVrg-CAG-GFP virus available with a Cre insert. Addgene has now updated its inventory and the AAVrg pmSyn1-EBFP-Cre is no longer available and so we are unable to perform surgeries in WT animals at this time as suggested by the reviewers.

Overall, the finding of GR knockout in the prelimbic area affecting behavior is mostly confirmatory with published work while providing some interesting phenomenological observations for future studies. As such, this is a methods paper describing a transgenic rat with much potential and may be better in a specialized journal or methods section of neuroscience journal.

The conditional knockout of GR specifically in the CaMKIIα positive neurons in the prelimbic cortex, as opposed to the entire mPFC, is novel and has not been described elsewhere. The reviewer is correct in that, our lab and others have previously shown behavioral and hormonal consequences of mPFC GR knockdown; however, these studies did not include females, nor did they have a specific promotor driven knockdown (meaning all neurons were affected unlike our CAMKIIα selective neuronal population). Additionally, we and others have also employed Cre-Driver lines to knockout GR but these genetic models are confounded by the developmental and peripheral functions of GR. We believe the current dataset is a more targeted approach in terms of cellular specificity, brain region targeted and time constrained and adds to the growing body of literature. Further, to our knowledge, differences in diving behavior have not been published after GRKD manipulations.

Reviewer #2:

Scheimann et al. generated a conditional GR knockout in rats using CRISPR/Cas9 technology. They provide evidence of targeted GR deletion in specific cell types, brain regions, and circuits via viral vector-mediated delivery of Cre. They also report that deletion of GR in output neurons of the prelimbic cortex resulted in sex-specific deficits in extinction of conditioned fear in females and a shift to active coping in the forced swim test in males. The manuscript is well-written and development of a conditional GR knockout model in rat is important and will be useful tool for probing GR function in the brain. However, there are several major limitations to the data that should be addressed to strengthen the conclusions of the manuscript.

The authors appreciate this positive feedback and have addressed the specific concerns below.

1) Given the sex-specific effects on behavior, it is critical to show and evaluate the immunostaining patterns from both male and female rats. Are sex-specific differences observed in the distribution, efficiency, and/or extent of GR knockout in male and female brains?

These additional experiments have now been performed and added to the paper (Results subsection “Behavioral Consequences of Targeted GR Deletion; Implications for Fear and Coping Behaviors”; Figure 3; Materials and methods paragraph three of subsection “Immunohistochemistry”). We found significant knockdown in both females and males and no overt sex differences between groups.

2) Does the loss of GR lead to compensatory increases in the expression of the related receptor MR? This is an important consideration given that MR binds corticosterone, is co-expressed with GR in many brain regions, and is known to play a role in stress and emotional behavior.

We did not assess MR expression in the current study and although MR and GR both bind corticosterone we are particularly interested in the role of GR as it plays a stronger role in the “stress corticosterone state” and subsequent behavioral consequences such as fear and coping strategies. Although MR and GR are somewhat similar we do not believe that MR acts in a direct compensatory capacity in response to GR knockout. This is beyond the scope of the current work but may be addressed in future studies.

3) Several issues should be addressed concerning Figure 2:

a) Why is the immunostaining pattern for GR in panel C different than in panel A? For the GR knockout cells in panel C, it appears that GR immunostaining is lost in the nucleus but not the cytoplasm. Why is this the case?

The immunostaining patterns appear different as 2 different GR antibodies were used. Panels A,B, E, F were all performed using a Santa Cruz (sc 1004) GR antibody prior to it being discontinued. Panels C and D were performed using an Invitrogen (PA1-511A) GR antibody. Both antibodies were rabbit polyclonal however they are not identical and thus have different immunostaining patterns.

b) The authors use AAVrg-CAG-GFP injection of SD:nr3c1fl/fl rats as a control for the AAVrg pmSyn1-EBFP-Cre injection (panels E and F). Another important control is injection of AAVrg pmSyn1-EBFP-Cre into wild-type rats. Was this control performed?

Addressed previously in response to reviewer #1.

“The authors acknowledge that the use of these 2 viruses may not be entirely clear. The viruses used for these experiments were sourced from Addgene. At the time these experiments were performed, this combination of “control” and “Cre” viruses were the best available. The AAVrg pmSyn1-EBFP-Cre virus was not available in a non-Cre construct nor was the AAVrg-CAG-GFP virus available with a Cre insert. Addgene has now updated its inventory and the AAVrg pmSyn1-EBFP-Cre is no longer available and so we are unable to perform surgeries in WT animals at this time as suggested by the reviewers.”

c) The images should be enlarged. The small size of the images makes it very difficult to evaluate the staining patterns.

All figures have now been uploaded as a separate files for clearer visualization.

4) Does knockout of GR in the prelimbic cortex alter locomotor activity of the rats?

We did not perform a separate measure of locomotion, such as the open field, in the current study.

5) The sample size for the GR knockout rats is quite small (n = 5 for females and n = 6 for males) for behavioral studies. In addition, a Fisher's LSD test was employed for post-hoc analyses. Are similar significance results found when statistical tests are utilized that correct for multiple comparisons?

We have re-ran our statistical analyses using Bonferroni post hoc to correct for multiple comparisons and found that all post hoc differences remain significant at p =/<0.05.

Reviewer #3:

This is the first study that develops a rat model with the capacity to perform cell- and region-specific deletion of the glucocorticoid receptor (GR) in rats. In addition to the novel tool development, the authors provided evidence of the behavioral impact of specific deletion of GR in the rat prelimbic CamKII-(glutamatergic) containing neurons. The data indicates differential effects of the knockdown approach on fear conditioning and extinction and behavior in the forced swim test in males and females, with a minor effect on corticosterone recovery in females. The study seems to be well performed and it is clearly written. Rats as model organisms have proved superior to mice for the dissection of stress (neuro)physiology and behavior and the current resource is a major step forward for the interrogation of GR function in this animal species.

The authors appreciate this positive feedback.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Please remove the statement concerning reviewer 1 below, and make certain the second issue raised is clearly noted as a caveat of the study if more data cannot be provided to address this directly.

Reviewer #1:

Below in quotes are the authors' responses to previous concerns. A follow up response is given. All other concerns were adequately addressed.

Authors: "…addition, we would like to clarify that we described the fact "the offspring of rat #60 can be bred to homozygosity indicates the correct targeting of loxP sequences to the Nr3c1 gene, instead of random integration". We did not mention anywhere in the text that offspring fecundity is evidence of no random integration."

My response: Although it is true the authors don't use the same verbiage, the ability to produce homozygous offspring should not be used as an indicator of correct targeting. The integration could still have additional integrants which would likely would be outcrossed if on a different chromosome or at least a sufficient distance from the targeted allele being genotyped. However, the only way to verify is to assay for such events and the authors have now done a more rigorous analysis to characterize the transgene integration. The statement in quotes provides an unnecessary precedent and contributes to the propagation of inadequate characterization of novel transgenic animals. It is not needed especially with the additional data and should be revised as simple statement of fact without indication or just remove it.

We appreciate the reviewer’s comment and have now changed the text accordingly to remove any indication. “We have consistently bred the offspring of rat #60 to homozygosity (Figure 1G).”

Authors: "The authors acknowledge that the use of these 2 viruses may not be entirely clear. The viruses used for these experiments were sourced from Addgene. At the time these experiments were performed, this combination of "control" and "Cre" viruses were the best available. The AAVrg pmSyn1-EBFP-Cre virus was not available in a non-Cre construct nor was the AAVrg-CAG-GFP virus available with a Cre insert. Addgene has now updated its inventory and the AAVrg pmSyn1-EBFP-Cre is no longer available and so we are unable to perform surgeries in WT animals at this time as suggested by the reviewers."

My response: Given that GFP expression has been demonstrated to cause toxicity, the concern is that any differences observed may be due to differences in viral transgene expression. Do AAVrg-CAG-GFP injected animals have same behavior as uninjected animals? At minimum, a strong emphasis must be placed on this caveat of the study. This is the only major concern remaining.

We appreciate the reviewer’s comment and have now clearly acknowledged this caveat in the text (Results subsection “Validation of Conditional Gene Deletion: Viral Vector Targeting”). “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 specific animals were used for immunohistochemical analysis only and were not used in any behavioral testing so we are unable to say at this time if the GFP-virus led to changes in behavior.

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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