Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide that acts on various cell populations in the brain and elicits stress-related behavioral (e.g. anxiety) and physiological (e.g. sympathomimetic) responses (Vale et al., 1981; Henckens et al., 2016). In the hypothalamic-pituitary-adrenal (HPA) axis, CRF released by neurons in the paraventricular nucleus of the hypothalamus (PVN) into the hypophyseal portal system stimulates adrenocorticotropic hormone (ACTH) release from the pituitary, which in turn stimulates release of glucocorticoids from the adrenal cortex. Glucocorticoids released into the circulatory system serve as effectors of the HPA axis by modulating various physiological (e.g. cardiovascular, respiratory, and immune) functions (Smith and Vale, 2006). Extrahypothalamic CRF neurons are located in many brain areas that modulate affective states and behaviors, such as the amygdala, cortex, hippocampus, midbrain, and locus coeruleus (Peng et al., 2017). Among these brain areas, the extended amygdala, particularly the central amygdala (CeA) and bed nucleus of stria terminalis (BNST), contain the highest densities of CRF neurons (Peng et al., 2017).

CRF type-1 (CRF₁) receptors are G_s- or G_q-coupled metabotropic receptors (Milan-Lobo et al., 2009; Wanat et al., 2008) that are highly expressed in brain areas that contain high densities of CRF fibers and/or CRF neurons, including the CeA, BNST, and medial amygdala. CRF signaling via CRF₁ modulates neurophysiological processes underlying stress reactivity, anxiety-related behaviors, learning and memory processes including fear acquisition and/or expression, pain signaling, and addiction-related behaviors (see reviews by Dedic et al., 2018; Henckens et al., 2016). Therefore, elucidation of the circuit-specific and cell-specific mechanisms by which CRF₁ signaling mediates these processes represents an important avenue of research for understanding behaviors related to stress, anxiety, fear, pain, and addiction.

The central amygdala (CeA) is a key nucleus in modulating affective states and behaviors related to stress, anxiety, fear, pain, and addiction (Gilpin et al., 2015). Functionally, the CeA serves as a major output nucleus of the amygdala and can be anatomically divided into lateral (CeAl) and medial (CeAm) subdivisions (Duvarci and Pare, 2014). In mice and rats, CRF-expressing (CRF⁺) neurons are densely localized to the CeAl, whereas CRF₁-expressing (CRF₁⁺) neurons are mostly localized to the CeAm (Day et al., 1999; Jolkkonen and Pitkänen, 1998; Justice et al., 2008; Pomrenze et al., 2015). In addition to receiving CRF input from CRF⁺ neurons in the CeAl, the CeAm receives CRF inputs from distal brain areas such as the bed nucleus of stria terminalis (BNST) (Dabrowska et al., 2016) and dorsal raphe nucleus (Commons et al., 2003). CeA CRF₁ signaling plays an important role in modulating affective states and behaviors. For example, pharmacological blockade of CeA CRF₁ attenuates stress-induced increases in anxiety-like behavior (Henry et al., 2006), nociception (Itoga et al., 2016), and alcohol drinking (Roberto et al., 2010; Weera et al., 2020), as well as fear acquisition and expression (Sanford et al., 2017). Work using transgenic CRF and CRF₁ reporter and Cre-driver mice have shed light on circuit- and cell-specific mechanisms by which CeA CRF⁺ and CRF₁⁺ cells mediate affective behaviors (e.g. Fadok et al., 2017; Sanford et al., 2017). However, mice have a limited behavioral repertoire when compared to rats, making the study of more complex behaviors (e.g. drug self-administration and social interaction; Homberg et al., 2017) difficult.

Here, we describe the generation of a novel CRF₁-Cre-^tdTomato transgenic rat line. We show that Crhr1 and iCre mRNA are expressed in the same neurons in the CeA and BLA using hybridization histochemistry. In the CeA, we show that ^tdTomato⁺ neurons are abundant in the CeAm, and that they are surrounded and in contact with CRF-containing puncta. We recorded membrane properties, inhibitory synaptic transmission, and spontaneous firing in CeA CRF₁-Cre-^tdTomato cells and show that these cells are sensitive to CRF. In addition, we show that Cre-dependent DREADD receptors can be targeted for expression by CeA CRF₁ cells such that DREADD stimulation of CRF₁⁺ CeA neurons increases nociception and anxiety-like behaviors, recapitulating prior work using pharmacological strategies. Outside the amygdala, we analyzed the expression of CRF₁-Cre-^tdTomato cells in several brain areas across the rostrocaudal axis and show that these cells are found in brain areas known to contain CRF₁-expressing cells. These anatomical, electrophysiological, and functional data support the utility of this CRF₁-Cre-^tdTomato transgenic rat line for the study of CRF₁ neural circuit function, and will be an important new resource that will complement CRFR1:GFP and CRFR1:Cre mice (Justice et al., 2008; Jiang et al., 2018; Sanford et al., 2017), and CRF-Cre rats (Pomrenze et al., 2015) for the study of CRF signaling in physiology and behavior.

We generated a new transgenic CRF₁-Cre-^tdTomato rat line to allow genetic manipulation and visualization of neurons that express CRF₁ receptors in the rat brain. We report that, within the CeA of CRF₁-Cre-^tdTomato rats, CRF₁-Cre-^tdTomato cells are located in the medial subdivision (CeAm), consistent with previous reports of Crhr1 expression in rats (Potter et al., 1994; Van Pett et al., 2000; Day et al., 1999) and mice (Van Pett et al., 2000; Justice et al., 2008), and that there is strong concordance ( > 90%) between Crhr1 and iCre mRNA expression in the CeAm of male and female CRF₁-Cre-^tdTomato rats. We also characterized the basal membrane properties, inhibitory synaptic transmission, and validated the CRF sensitivity of ^tdTomato-expressing CeA CRF₁⁺ cells in male and female rats. In addition, we showed that stimulatory DREADD receptors [hM3D(Gq)] can be targeted to CeA CRF₁-Cre-^tdTomato cells using a Cre-dependent expression strategy, that systemic CNO treatment induces c-Fos in hM3D(Gq)-transduced CRF₁⁺ cells in CeA, and that CNO induces membrane depolarization and spontaneous firing activity in hM3D(Gq)-transduced CRF₁⁺ cells in CeA. We showed that hM3D(Gq)-mediated stimulation of CeA CRF₁⁺ cells increases anxiety-like behavior, as measured by EPM and open field tests, as well as mechanical nociception as measured by the Von Frey test. Finally, we report that Crhr1 and iCre mRNA expression are highly colocalized in the BLA, and that Cre-^tdTomato is expressed in several brain areas across the rostrocaudal axis of the rat brain in expected patterns. Collectively, this work provides cellular, electrophysiological, and behavioral data demonstrating the validity and reliability of a new transgenic rat model for the identification and selective manipulation of CRF₁⁺ neurons in the CeA.

CRF₁-Cre-^tdTomato rats were generated by modifying a BAC clone derived from F344 rats, that was injected into oocytes derived from Wistar rats. Because the originating strain of the BAC DNA differs from the strain used for transgenesis, DNA sequence differences between F344 and Wistar rats in the 200 + kb region surrounding the Crhr1 genomic locus may impact expression of iCre-2A-^tdTomato in the CRF₁-Cre transgenic rat. Moreover, because transgenic BAC DNA insertion was not directed, position effect, copy number, and fragmentation of the BAC insertion may possibly alter expression of reporter genes. These sources of variability were minimized by the expertise of the UNC Transgenic Core Facility, where BAC DNA was prepared, cleaved at a single site to produce full-length single stranded BAC DNA, then injected into oocytes. Two transgenic founders were isolated that contained unique DNA sequences near the Crhr1 genomic locus in the BAC, as well as DNA sequence from both terminal ends of the injected BAC, indicating insertion of full-length BAC DNA. Of these two rats, one displayed expression of ^tdTomato in a pattern reflecting Crhr1 expression patterns reported in rats (Potter et al., 1994; Van Pett et al., 2000), as well as transgenic expression of reporter genes reported in similarly designed BAC transgenic mice (Justice et al., 2008; Jiang et al., 2018; Hunt et al., 2018). The genomic locus and copy number of transgenic BAC insertion have not been further characterized. However, the high degree of alignment of Cre-^tdTomato expression with known CRF₁ expression patterns broadly throughout the brain suggests that position, copy number, or fragmentation did not substantially alter expression of the transgene. In addition, the behavioral and physiological phenotypes of CRF₁-Cre-^tdTomato rats reported here closely resemble those seen in wild-type Wistar rats (e.g., Itoga et al., 2016; Albrechet-Souza et al., 2020; see below), suggesting that BAC insertion did not produce observable anomalous phenotypes.

The intrinsic properties of CRF₁⁺ (^tdTomato⁺) CeA neurons in CRF₁-Cre-^tdTomato rats are similar to those reported in wild-type rat CeA neurons (Herman and Roberto, 2016), suggesting that the transgene did not significantly impact the overall health or activity of CRF₁⁺ CeA neurons. Previous studies have employed a CRFR1:GFP transgenic mouse model to examine CRF₁⁺ CeA neurons in local CeA microcircuitry (Herman et al., 2013; Herman et al., 2016). Although prior work was conducted in mouse and some species differences would be expected, the electrophysiological properties of CRF₁⁺ neurons in the CeA are relatively consistent between the mouse and rat transgenic models. Recent work specifically examining sex differences in CRF₁⁺ neurons from CRFR1:GFP mice reported similar intrinsic membrane properties, cell-typing, and baseline inhibitory transmission as reported here and noted no sex differences in basal properties between male and female CRF₁⁺ neurons in the CeA (Agoglia et al., 2020), consistent with our current findings. In contrast to previous work, however, we found no sex differences in CRF sensitivity of CRF₁⁺ cells in CeA. Although CRF was previously found to increase firing in CRF₁⁺ CeA neurons in both male and female mice, CRF₁⁺ CeA neurons from male mice displayed a significantly greater increase in firing in response to CRF application (Agoglia et al., 2020). This discrepancy may be driven by differences in sampling size (although the current study was not sufficiently powered to detect sex differences), high variability in male baseline firing rates observed here, sex differences in baseline firing rates observed here, or it may reflect a species difference in sex-dependent sensitivity to CRF. Additional studies are required for a more comprehensive examination of sex- and/or species-specific differences in CRF-stimulated CRFR1⁺ neuronal activity in distinct brain regions. CRF₁⁺ neurons in the CeA have also previously been implicated in the neuroplastic changes associated with acute and chronic ethanol exposure in mice (Herman et al., 2013; Herman et al., 2016), and future work will determine if this is also the case in rats.

To test the feasibility of using Cre-dependent stimulatory DREADDs to interrogate the role of CeA CRF₁⁺ cells in behavior, we targeted Gq-coupled DREADD receptors [DIO-hM3D(Gq)] to CeA CRF₁-Cre cells and first tested the effects of CeA CRF₁⁺ cell stimulation on nociception. We showed that DREADD stimulation of CeA CRF₁⁺ cells increases mechanical sensitivity as measured by the Von Frey test, but not thermal sensitivity measured by the Hargreaves test. Numerous studies have implicated CeA CRF-CRF₁ signaling in nociception. For instance, pain induced by carrageenan injection into the knee joint produces hyperactivity of CeA neurons in rats, a phenomenon that is CRF₁ dependent (Ji and Neugebauer, 2007). Conversely, latency for hind limb withdrawal reflex induced by knee joint pressure application is decreased (reflecting hyperalgesia) by CRF infusion into the CeA, a phenomenon that is blocked by co-infusion of a CRF₁ antagonist (Ji et al., 2013). With regard to mechanical sensitivity, our finding that DREADD activation of CeA CRF₁ neurons produces mechanical hypersensitivity extends previous work showing that ablation or inhibition of CeA CRF neurons blocks neuropathic pain-induced mechanical hypersensitivity, as measured by the Von Frey test (Andreoli et al., 2017). Various studies have also previously reported that CeA CRF₁ antagonism attenuates mechanical hypersensitivity produced by chronic drug or alcohol exposure (e.g. Cohen et al., 2015; Edwards et al., 2012). Collectively, this prior work suggests that activation of CeA CRF-CRF₁ signaling by chronic injuries or insults facilitates a hyperalgesic state, which was recapitulated in the current study via chemogenetic stimulation of CeA CRF₁ cells. It should be noted that chemogenetic stimulation of CeA CRF⁺ cells has been shown to potentiate stress-induced analgesia (Andreoli et al., 2017), although this effect may be due to CRF release outside the CeA (e.g. locus coeruleus) or due to changes in GABA release from CRF⁺ cells. Within the CeA, pharmacological studies suggest that pro- and anti-nociceptive effects of CRF signaling may be mediated by CRF₁ and CRF₂ receptors, respectively (Ji and Neugebauer, 2007; Ji and Neugebauer, 2008), but further studies are required to delineate the precise mechanisms by which CeA CRF signaling supports hyperalgesia and analgesia. The CRF₁-Cre-^tdTomato rat described here represents a useful tool for these studies.

Our lab has shown that predator odor stress-induced hyperalgesia, as measured by the Hargreaves test, is mediated by increased CRF-CRF₁ signaling in the CeA (Itoga et al., 2016). Contrary to our hypothesis, we did not observe any effect of DREADD activation of CeA CRF₁ cells on thermal sensitivity. It is not clear why CRF₁-Cre rats exhibited mechanical hypersensitivity but not thermal hyperalgesia in this study. It is possible that specific CeA cell populations are involved in specific types of nociceptive processing, or it is possible that the engagement/recruitment of CeA CRF₁⁺ cells in mediating specific types of nociception depends on the animal’s history or affective state. Using the CRF₁-Cre-^tdTomato rat line reported here, future work will elucidate the role of specific CRF₁⁺ circuits in mechanical and thermal sensitivity (which may or may not be partially overlapping) under basal and challenged conditions such as neuropathic or inflammatory pain, stress exposure and/or withdrawal from chronic exposure to drugs or alcohol.

CeA CRF-CRF₁ signaling generally promotes anxiogenic responses, particularly under challenged conditions such as stress or withdrawal from chronic exposure to drugs of abuse. For instance, in mice, intra-CeA infusion of a CRF₁ antagonist attenuates anxiety-like behavior, as measured by open field and light-dark box tests, following immobilization stress but not under basal conditions (Henry et al., 2006). Similarly, blockade of CRF-CRF₁ signaling in the CeA attenuates alcohol withdrawal-induced anxiety in rats, as measured by the EPM test (Rassnick et al., 1993). Exposure to stressors (Merlo Pich et al., 1995) and alcohol withdrawal (Zorrilla et al., 2001) both increase extracellular CRF levels in the CeA. Messing and colleagues used CRF-Cre rats to show that CeA CRF cell activation (using DREADDs) increases anxiety-like behavior and that this effect is blocked by CeA CRF knockdown using RNA interference (Pomrenze et al., 2019). Collectively, these studies show that increased CRF-CRF₁ signaling in CeA, whether produced by stress exposure, drug withdrawal, or the use of viral genetic tools, supports anxiogenesis. Here, we showed that chemogenetic stimulation of CeA CRF₁⁺ cells increases anxiety-like behavior, as measured by EPM and open field tests. Interestingly, we did not observe an effect of chemogenetic stimulation of CeA CRF₁⁺ cells on light-dark box measures, including time spent in the light vs. dark boxes, latency to enter light box, and number of crosses between the two compartments. Previous studies reported that the light-dark box test may be more suited for detecting anxiolytic rather than anxiogenic effects (Crawley and Davis, 1982), and that animals’ responses in this test is affected by the intensity of illumination in the animal’s regular housing room (File et al., 2004). Future studies will test if Cre-dependent expression of inhibitory chemogenetic or optogenetic strategies can be applied to inhibit CeA CRF₁ cells, and if inhibition of CeA CRF₁ cells reduces anxiety-like behavior at baseline or after stress exposure.

Although the CeA is generally not thought to exhibit strong sexual dimorphism, sex differences have been reported for CRF and CRF₁ properties in the CeA. For example, female rats in proestrus have higher levels of CeA Crf mRNA than male rats and footshock stress induces greater CeA Crf expression in female proestrus than in male rats (Iwasaki-Sekino et al., 2009). However, using autoradiography, Cooke and colleagues reported no sex differences in CRF₁ receptor binding in the CeA of rats (Weathington et al., 2014). Using a transgenic CRFR1:GFP mouse line, the CeA of male animals was shown to contain more CRF₁⁺ cells than female animals (Agoglia et al., 2020). In the current study, although underpowered to detect sex effects, we found that male CRF₁-Cre-^tdTomato rats tend to have more CRF1⁺ cells within the CeA than female rats, as detected via RNAscope in situ hybridization. Previous work using CRFR1:GFP mice (Agoglia et al., 2020) and our current work using CRF₁-Cre-^tdTomato rats revealed no sex differences in basal membrane properties and inhibitory synaptic transmission in CeA CRF₁⁺ neurons. Our previous work in CRFR1:GFP mice showed that, while CRF application increased firing in CRF₁⁺ CeA neurons from both female and male mice, larger increases were observed in CRF₁⁺ neurons from male mice, an effect that we did not observe in rats potentially due to differences in variability or sex differences in firing rate.

The overwhelming majority of published studies examining the role of CeA CRF₁ signaling in nocifensive and anxiety-like behaviors has employed only male subjects. Here, we used both sexes in tests of nociception and anxiety-like behavior and we did not detect any sex differences. However, we observed that the anxiogenic effect of CeA CRF₁⁺ cell activation in the EPM test was driven by stronger effects in male subjects. One interpretation is that CeA CRF₁⁺ cell activation affects specific components of anxiety-related behavior, that these components are aligned with specific ‘anxiety-like behavior’ phenotypes in male versus female rats that are more or less detectable by these various tests. In support of this idea, a meta-analysis of studies of anxiety-like behavior in rats and mice revealed a discordance in results between the EPM and open field tests (Mohammad et al., 2016). However, due to the small number of studies testing female animals, the role of sex in this discordance is still unknown. Within the EPM literature, studies have shown that the EPM test is more reliable for detecting anxiogenic effects in male rather than female rodents (e.g. Scholl et al., 2019). Collectively, these findings demonstrate the importance of behavioral assay selection and of using both male and female subjects.

In summary, we present a novel CRF₁-Cre-^tdTomato rat line that allows for the visualization and manipulation of CRF₁-expressing cells. The CRF₁-Cre rat can be used to study the role of distinct CRF₁⁺ cell populations and circuits in physiology and behavior. In addition, the expression of the fluorescent reporter ^tdTomato in CRF₁-expressing cells will allow for high resolution, detailed analysis of the expression pattern of CRF₁ that has not been possible due to the lack of CRF₁-specific antibodies.

All data generated during this study are included in the manuscript.

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Author details

1. Marcus M Weera

   Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review and editing

   For correspondence

   mweera@lsuhsc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2451-0350

2. Abigail E Agoglia

   Department of Pharmacology, University of North Carolina, Chapel Hill, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

3. Eliza Douglass

   Department of Pharmacology, University of North Carolina, Chapel Hill, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

4. Zhiying Jiang

   Institute of Molecular Medicine, University of Texas Health Sciences Center, Houston, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Shivakumar Rajamanickam

   Institute of Molecular Medicine, University of Texas Health Sciences Center, Houston, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Rosetta S Shackett

   Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

7. Melissa A Herman

   1. Department of Pharmacology, University of North Carolina, Chapel Hill, United States
   2. Bowles Center for Alcohol Studies, University of North Carolina, Chapel Hill, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Nicholas J Justice and Nicholas W Gilpin

   Competing interests

   No competing interests declared

8. Nicholas J Justice

   1. Institute of Molecular Medicine, University of Texas Health Sciences Center, Houston, United States
   2. Department of Integrative Biology and Pharmacology, McGovern Medical School at UT Health, Houston, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Writing – review and editing

   Contributed equally with

   Melissa A Herman and Nicholas W Gilpin

   Competing interests

   No competing interests declared

9. Nicholas W Gilpin

   1. Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, United States
   2. Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, United States
   3. Alcohol & Drug Abuse Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, United States
   4. Southeast Louisiana VA Healthcare System (SLVHCS), New Orleans, United States

   Contribution

   Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing

   Contributed equally with

   Melissa A Herman and Nicholas J Justice

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8901-8917

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