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. Author manuscript; available in PMC: 2026 Apr 22.
Published in final edited form as: Addict Neurosci. 2026 Feb 11;19:100257. doi: 10.1016/j.addicn.2026.100257

The central amygdala corticotropin releasing factor system regulates anxiety-like behavior in an age- and sex- dependent manner in rats

Kelcie C Schatz a,b, Margaret Agajanov a,b, Elena I Varlinskaya a,b, Marvin R Diaz a,b
PMCID: PMC13089615  NIHMSID: NIHMS2162132  PMID: 42006257

Abstract

The corticotropin releasing factor (CRF) system is a key regulator of anxiety-like behavior and a major contributor to addiction. The canonical anxiogenic role of CRF has been largely based on CRF manipulations in adult male rodents, particularly within the central amygdala (CeA), despite evidence that underlying neurobiological mechanisms of anxiety are sex- and age-dependent. Our lab has shown that the physiological response to CRF receptor 1 (CRFR1) activation within the medial CeA, a brain region associated with anxiety and addiction, varies with both age and sex. Therefore, in the current study, we investigated the effects of CRFR1 activation in the CeA on anxiety-like behavior in naïve juvenile (~postnatal day [P] 25), adolescent (~P45), or adult (~P80) male and female Sprague Dawley rats. Rats were given bilateral cannula in the CeA and then tested in the light-dark box test following an infusion of either the CRFR1 agonist Stressin-1 (1 μM) or vehicle. In contrast to the previously established anxiogenic effects of CRF agonists, we found that intra-CeA Stressin-1 decreased anxiety-like behavior in females regardless of age and juvenile males, with no significant effects evident in adolescent or adult males. Notably, RNAscope in situ hybridization revealed no differences in CRFR1 or CRF mRNA across age or sex. Taken together, these findings further highlight the importance of age and sex on CRF system regulation of anxiety-like behavior, providing a background for the development of more targeted treatments of anxiety disorders and addiction.

Keywords: corticotropin releasing factor, central amygdala, age differences, sex differences, anxiety

Introduction

Anxiety disorders are the most prevalent mental health conditions worldwide (World Health Organization, 2023). It is estimated that nearly 30% of people will experience an anxiety disorder in their lifetime, although this prevalence is likely underestimated due to frequent underreporting and lack of treatment (Bandelow & Michaelis, 2015; Momin et al., 2023). Anxiety disorders can emerge at any age, from early childhood to adulthood, and there are sex differences in the age of onset and symptom presentation (Altemus et al., 2014; Kessler et al., 2007; Kessler et al., 2009; Lewinsohn et al., 1998; Merikangas et al., 2010). Since the emergence of anxiety symptoms early in life is a risk factor for developing mood disorders and addiction across the lifespan, it is critical to investigate the neural mechanisms underlying anxiety across development and in both sexes (Beesdo et al., 2009; Doering et al., 2022; Doering et al., 2019). A more precise understanding of the neural mechanisms that underlie anxiety across age and sex will help inform more targeted treatment development to better address anxiety disorder symptom management within specific populations.

Corticotropin releasing factor (CRF) is a powerful regulator of stress and anxiety-like behavior through its control of the hypothalamic pituitary adrenal (HPA) axis as well as its extensive extra-hypothalamic projections. Traditionally, CRF has been considered to be anxiogenic, with intracerebroventricular (ICV) infusion of CRF increasing anxiety-like behavior, and CRF receptor 1 (CRFR1) knock out mice displaying decreased anxiety-like behavior (Baldwin et al., 1991; Smith et al., 1998; Spina et al., 2002; Timpl et al., 1998). CRF-expressing neurons and CRFR1 are highly expressed in the central amygdala (CeA), a brain region critically involved in the response to anxiogenic stimuli (Agoglia & Herman, 2018; Gilpin et al., 2015; Hare et al., 2008; Liberzon & Sripada, 2008). Blockade of CeA CRFRs ameliorates the anxiogenic effects of ethanol withdrawal, and chemogenetic stimulation of CRF or CRFR1 containing CeA cells increases anxiety-like behavior, supporting a role for the CeA CRF system in anxiety-like behavior (Pomrenze et al., 2019; Rassnick et al., 1993; Weera et al., 2022). While children and adolescents of both sexes are at heightened risk for the development of anxiety disorders, most preclinical evidence linking CeA activation to increased anxiety comes from studies in adult male laboratory rodents.

The CeA is thought to regulate anxiety-like behavior, in part, through its GABAergic projections to downstream targets (Babaev et al., 2018). In adult males, CRFR1 activation enhances CeA GABA transmission, a mechanism that likely underlies the canonical anxiogenic role of CeA CRFR1 signaling (Babaev et al., 2018). In contrast, CeA CRFR1 activation reduces GABA transmission in adolescent males; notably, this age-dependent neurophysiological switch is not evident in females, as CeA CRFR1 activation decreases GABA transmission in both adolescent and adult females (Rouzer & Diaz, 2021). These findings suggest that CeA CRFR1 activation may regulate anxiety-like behavior in an age- and sex-specific manner – promoting anxiety-like behavior in adult males, but reducing it in adolescent males and females at both ages. However, investigation of the effect of CeA CRFR1 activation on anxiety-like behavior has been limited to adult males and has not yet been explored in females or in younger males. Therefore, the current study tested the effect of bilateral CeA CRFR1 activation on anxiety-like behavior in juvenile, adolescent, and adult male and female rats, and determined the expression of CRF and CRFR1 within the CeA across ontogeny. A clear understanding of the complex mechanisms that underlie anxiety throughout development is essential for advancing more precise and targeted treatments for anxiety disorders.

Methods

Subjects

Sprague Dawley rats were bred in house using male and female breeders obtained from Envigo (Indianapolis, IN, USA). Pregnancy was determined by sperm detection in a vaginal sample. The subjects used in these experiments were taken from 35 litters. Litters were culled to 8-12 pups on postnatal day (P)2, with most litters containing 12 pups. A 1:1 male to female ratio was maintained whenever possible. Pups were weaned on P20 and pair-housed with same-sex littermates. All rats were provided with a plastic hut and crinkle paper enrichment at the time of weaning, which remained available to them until their day of surgery. Following recovery from surgery, all rats were put back with their original cage mate and were provided with crinkle paper enrichment only. All animals were maintained on a 12:12h light:dark cycle in a temperature-controlled vivarium with ad libitum access to food (Purina Lab Diet 5008C33) and water. All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at Binghamton University.

Stereotaxic Surgeries and Microinfusions

Stereotaxic surgeries were performed between P21-25 (juvenile), P40-45 (adolescent), and P75-80 (adult). Rats were anesthetized with isoflurane (3% induction, 1-2% maintenance) and placed in the stereotaxic frame for bilateral implantation of 23-gauge stainless steel guide cannulae (P1 Technologies, Roanoke, VA, USA) aimed at the CeA using the following coordinates – juveniles: −1.5 mm posterior to bregma, ± 3.9 mm from midline, −6.0 mm ventral from skull; adolescents: −1.7 mm posterior to bregma, ± 4.1 mm from midline, −6.4 mm ventral from skull; adults: −2.0 mm posterior to bregma, ± 4.1 mm from midline, −7.0 mm ventral from skull. Guide cannulae were secured to the skull using anchor screws and dental cement. Following recovery from anesthesia, rats were re-housed with their original cage mate until testing began. Subjects received postoperative intraperitoneal injections of buprenorphine (0.03 mg/kg) every 12 hours for 48 hours following surgery and subjects were weighed and handled daily after surgery to familiarize them with the drug microinfusion procedure.

Following a 5-6 day post-surgical recovery period, each rat received either a vehicle (oxygenated [95% oxygen, 5% carbon dioxide] artificial cerebrospinal fluid [ACSF]: 125 mM NaCl, 2 mM KCl, 1.3 mM NaH2PO4, 26 mM NAHCO3, 10 mM glucose, 2 mM CaCl2, 1mM MgSO4, 0.4 mM ascorbic acid) or 1 μM Stressin-1 (Tocris, Minneapolis, MN, USA) bilateral infusion. Rats received 0.25 μl per side over 1 min using a Hamilton syringe and microinfusion pump (Stoelting, Wood Dale, IL, USA). Injectors were left in place for 1 min after the infusion. Microinfusions and behavior testing were conducted 2-5 hours after lights on.

Light-Dark Box (LDB)

Anxiety-like behavior was tested in the LDB 10 min after the microinfusions. Briefly, subjects were placed in the light chamber of the box, facing away from the dark chamber of the box, and were allowed to explore the apparatus freely for 15 minutes under dim light (~15 lux). Tests were video recorded for later scoring of time spent in the light chamber, latency to exit the light chamber and enter the dark chamber (egress latency), and the number of head pokes from the dark chamber into the light chamber. Videos were scored by an individual blind to the experimental group. Apparatus was disinfected with 3% hydrogen peroxide and allowed to completely dry between subjects. All tests were conducted by one female experimenter who was blinded to all experimental conditions. Following completion of the LDB test, subjects were euthanized, ink was infused through the guide cannula, and brains were extracted for subsequent verification of cannula placements. Only subjects that had correctly positioned bilateral cannula in the CeA were included in the final experimental analyses. Supplementary Figure 1 depicts where the drug infusions were placed in the CeA (Swanson, 2004).

RNAscope in situ hybridization

In a different subset of non-behaviorally tested animals, juvenile, adolescent, and adult rats were anesthetized with isoflurane vapor (3-5%), decapitated, and brains were rapidly removed and flash frozen in 2-methylbutane. Brains were sectioned (14 μm thick) and immediately thaw mounted onto positively charged glass microscope slides. RNAscope multiplex fluorescent assays were performed as recommended by the manufacturer, Advanced Cell Diagnostics (ACD Bio, Newark, CA, USA), using protease III for 20 min to avoid over-digestion of samples. For each subject, 2 slices on the same slide were stained with riboprobes designed to target CRFR1 mRNA (ACD catalog# 31891) and CRF mRNA (ACD catalog# 318931). Positive (catalog #320881) and negative control (catalog #320891) probes were also utilized to verify the accuracy of the assay. All slides were counterstained with DAPI to label cell nuclei (included in ACD Multiplex Fluorescent Reagent Kit, catalog# 323100) and were subsequently cover-slipped using ProLong Gold Antifade Mountant (Thermo Fisher Scientific).

Slides were imaged at 40X magnification with an Olympus VS200 Slide Scanner using identical image acquisition settings. Images were analyzed using Halo (Indica Labs) with identical detection settings across all images. The regions of interest for analysis were designated for each image by placing a standardized ellipse (height: 400μm, width: 300μm) in the lateral subnucleus of the CeA (CeL) and another ellipse in the medial subnucleus of the CeA (CeM). Cells were detected using a 3 μm maximum radius around DAPI labeled nuclei. To reduce the possibility of false positives, cells were considered positive for CRFR1 or CRF if they contained at least 3 distinct transcripts for a probe. An average of all images analyzed for each subject was used as a single data point per subject in all analyses.

Statistical Analysis

All analyses were conducted using GraphPad Prism 9. For LDB test, the original sample sizes per group were: 22 juvenile females (11 aCSF, 11 Stressin-1), 17 juvenile males (7 aCSF, 10 Stressin-1), 25 adolescent females (15 aCSF, 10 Stressin-1), 19 adolescent males (9 aCSF, 10 Stressin-1), 26 adult females (11 aCSF, 10 Stressin-1), and 20 adult males (10 aCSF, 10 Stressin-1). All data were first assessed for outliers using the ROUT method, and outliers were removed from analyses. Each LDB measure was analyzed with a 3 (age) × 2 (sex) × 2 (drug) analysis of variance (ANOVA), followed by 3 (age) × 2 (drug) ANOVAs conducted separately for males and females. RNAscope data were analyzed using a 3 (age) x 2 (sex) ANOVA. The sample sizes per group for RNAscope analysis were: 5 juvenile females, 5 juvenile males, 6 adolescent females, 5 adolescent males, 6 adult females, and 5 adult males. When there was an outlier in number of total cells analyzed in RNAscope, that animal was removed from all subsequent analyses for that region. When main effects and interactions were detected, Tukey post-hoc tests were performed to explore these effects. Data are reported as mean ± SEM. Significance was assumed when p ≤ 0.05.

Results

CeA CRFR1 activation alters anxiety-like behavior in a sex-specific manner

Omnibus ANOVAs revealed significant main effects or interactions including sex for each measure. Specifically, total time spent in the light zone of the LDB differed as a function of sex [F(1, 108) = 6.884, p = 0.010], while an interaction of drug and sex [F(1, 107) = 5.768, p = 0.018] was evident for time spent in the light zone within the first 5 minutes of the 15-minute test. Analysis of the egress latency from the light zone to the dark zone also revealed a drug by sex interaction [F(1, 111) = 4.689, p = 0.033], and the number of head pokes from the dark chamber into the light chamber also differed as a function of sex [F(1, 113) = 9.587, p = 0.002]. Therefore, the LDB data were analyzed separately for males and females.

In general, Stressin-1 did not impact behavior in adolescent and adult males, with juvenile males showing some drug-induced changes. Specifically, total time spent in the light zone differed only as a function of age [F(1, 45) = 3.18, p = 0.050, Figure 1A], with adult males spending significantly more time in the light chamber than their adolescent counterparts (p = 0.041). There was no main effect of drug in males (p > 0.05), while age and drug interaction only trended towards significance [F(2, 45) = 2.64, p = 0.08]. Time spent in the light zone within the first 5 minutes of the 15-minute test, when the context was most novel, differed as a function of age in males [F(2, 48) = 4.82, p = 0.01, Figure 1B], with adult males spending more time in the light than adolescent males (p = 0.01). Stressin-1 did not affect this measure, with the interaction between age and drug also not reaching significance [F(2, 48) = 2.60, p = 0.08]. In males, egress latency differed as a function of age [F(2, 48) = 6.879, p = 0.002, Figure 1C], with adults having a longer egress latency than juveniles (p = 0.01) and adolescents (p = 0.004). In male subjects, number of head pokes was the only measure affected by Stressin-1. The ANOVA revealed an interaction of age by drug [F(1, 47) = 5.449, p = 0.007, Figure 1D], with Stressin-1 significantly increasing head pokes in juveniles (p = 0.040).

Figure 1.

Figure 1.

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Behavior of juvenile, adolescent and adult male rats in the LDB test following infusions of aCSF or Stressin-1 into the CeA. (A) Total time spent in the light chamber was significantly higher in adult males than their adolescent counterparts. (B) Time spent in the light chamber during the first 5 min of the test was significantly higher in adult males than their adolescent counterparts. (C) Egress latency was significantly longer in adult males than juveniles and adolescents. (D) Head pokes were significantly increased by Stressin-1 in juveniles, with no effects evident in older males. Age differences are marked with # (# - p < 0.05, ## - p < 0.01), with data collapsed across drug condition. Asterisk marks significant Stressin-1-induced changes (* - p < 0.05).

In contrast, female behavior in the LDB was affected by Stressin-1. Total time in the light zone (Figure 2A) differed as a function of age [F(2, 63 = 4.073, p = 0.02] and drug [F(1, 63) = 5.43, p = 0.022]. Juvenile females spent more time in the light chamber than adults, and Stressin-1 infusions in the CeA increased time spent in the light across all ages. In female subjects, Stressin-1 increased time in the light within the first 5 minutes of the test [F(1, 59) = 5.88, p = 0.01, Figure 2B), with no effect of age evident for this measure. Egress latency was also affected by drug [F(1, 63) = 4.51, p = 0.03, Figure 2C], with Stressin-1 increasing egress latency regardless of ages. Number of head pokes was the only behavioral measure not affected by Stressin-1 in female subjects (Figure 2D).

Figure 2.

Figure 2.

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Behavior of juvenile, adolescent and adult female rats in the LDB test following infusions of aCSF or Stressin-1 into the CeA. (A) Total time spent in the light chamber was significantly higher in juvenile females than in their adult counterparts, and Stressin-1 infusion significantly increased time in the light regardless of age. Stressin-1 also increased time in the light during the first 5 min of the test (B) and egress latency (C), with data collapsed across age. (D) Head pokes did not differ across age and were not affected by Stressin-1. Age differences are marked with # (p < 0.05), with data collapsed across drug condition. Asterisks mark significant Stressin-1-induced changes (* - p < 0.05) with data collapsed across age.

CeA CRFR1 mRNA does not differ between sexes or across development

Differences in CRFR1 and/or CRF expression within the CeA could potentially account for the observed age- and sex-dependent effects of CeA Stressin-1 microinfusions on anxiety-like behavior. Thus, CRFR1 and CRF mRNA was measured in the CeM and CeL using RNAscope (Figures 3A-B). The number of DAPI labeled cells analyzed in the CeM or the CeL were not different between the 3 ages [CeM: F(2, 26) = 0.69, p = 0.50; CeL: F(2, 26) = 0.16, p = 0.85], between the 2 sexes [CeM: F(1, 26) = 0.02, p = 0.88; CeL: F(1,26) = 0.01, p = 0.90], nor was there an interaction between age and sex [CeM: F(2, 26) = 0.27, p = 0.76; CeL: F(2, 26) = 0.20, p = 0.81]. Investigation of total number of CRF and CRFR1 transcripts or the %CRF+ and %CRFR1+ cells in the CeM or in the CeL showed no significant differences as a function of age or sex (Figures 3C-F); these detailed statistical analyses can be found in Table 1. Supplementary Figure 2 shows CRF and CRFR1 total transcript numbers in the CeL and CeM.

Figure 3.

Figure 3.

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Representative images of the RNAscope assay. Image of the amygdala was acquired at 40X on an Olympus VS200 Slide Scanner showing the overlay of the nuclear stain DAPI (blue), CRFR1 mRNA (green), and CRF mRNA (red) (A). Zoomed in images were acquired at 40X on Leica SP5 Laser Scanning Confocal Microscope showing the nuclear stain DAPI, CRFR1 mRNA, CRF mRNA, and the overlay (B). There were no differences in %CRFR1+ or %CRF+ between juvenile, adolescent, and adult male and female rats in the CeM (D, F) or the CeL (C,E). Representative images are from an adult male subject.

Table 1.

Statistics from RNAscope analysis of CRF and CRFR1 transcript and %CRF+ and %CRFR1+ cells in the CeM and CeL. There were no significant main effects of sex (second column), age (third column), or an interaction between the two (fourth column).

Sex Age Interaction
CeM
CRF transcript F(1,26) = 0.80
p = 0.37		 F(2,26) = 0.88
p = 0.42		 F(2,26) = 0.86
p = 0.43
%CRF+ F(1,26) = 0.004
p = 0.94		 F(2,26) = 0.38
p = 0.68		 F(2,26) = 0.31
p = 0.73
CRFR1 transcript F(1,26) = 0.30
p = 0.58		 F(2,26) = 1.16
p = 0.32		 F(2,26) = 2.23
p = 0.12
%CRFR1+ F(1,26) = 0.30
p = 0.09		 F(2,26) = 1.86
p = 0.17		 F(2,26) = 0.83
p = 0.44
CeL
CRF transcript F(1,26) = 0.57
p = 0.45		 F(2,26) = 0.54
p = 0.58		 F(2,26) = 0.32
p = 0.72
%CRF+ F(1,26) = 0.13
p = 0.71		 F(2,26) = 0.23
p = 0.79		 F(2,26) = 0.05
p = 0.94
CRFR1 transcript F(1,26) = 0.006
p = 0.93		 F(2,26) = 1.65
p = 0.21		 F(2,26) = 0.96
p = 0.39
%CRFR1+ F(1,26) = 0.38
p = 0.54		 F(2,26) = 0.86
p = 0.43		 F(2,26) = 1.21
p = 0.31
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Discussion

The CeA CRF system plays a crucial role in regulating stress, anxiety, and negative affect, which has made it a target of extensive investigation to determine how it can be targeted to alleviate symptoms of disorders characterized by anxiety such as generalized anxiety disorder, posttraumatic stress disorder, and substance use disorders (Kehne, 2007; Koob & Zorrilla, 2012; Valdez, 2006). Therefore, the current study determined the impact of CeA CRFR1 activation on anxiety-like behavior in male and female rats tested during the juvenile, adolescent, and adult developmental periods. We found that CeA CRFR1 activation with a 1 μM dose of Stressin-1 decreased anxiety-like behavior in females at all 3 ages, as evidenced by increased time in the light chamber and increased egress latency. However, the same dose of Stressin-1 did not elicit any significant behavioral changes in adolescent and adult males, with only juvenile males demonstrating increased number of head pokes. Furthermore, age or sex did not impact CRF or CRFR1 mRNA expression in the CeM or the CeL. Together, these findings highlight the complexity of the CRF system within the CeA and emphasize the necessity of considering both age and sex when evaluating its function (Agoglia et al., 2020; Agoglia & Herman, 2018; Rouzer & Diaz, 2021; Wolfe et al., 2019).

In contrast to the canonical anxiogenic role of CRF (Agoglia & Herman, 2018; Gilpin et al., 2015), CeA CRFR1 activation decreased female anxiety-like behavior in the light-dark box. Given that increased CeA inhibitory neurotransmission is related to increased anxiety-like behavior (Babaev et al., 2018), this result is in line with our previous electrophysiological finding indicating that CRFR1 activation, specifically within the CeM, decreases GABA release in adolescent and adult females (Rouzer & Diaz, 2021). However, in adolescent males, CeM CRFR1 activation decreases GABA release, whereas GABA release is increased in adult males following CeM CRFR1 activation, representing an age-dependent switch in CRFR1 function at the synaptic level in males (Nie et al., 2009; Roberto et al., 2010; Rouzer & Diaz, 2021, 2022). It is worth noting that although it failed to reach statistical significance (p = 0.08), in males there was a trend toward an interaction between age and drug in the amount of time spent in the light zone of the LDB, suggestive of an anxiogenic effect of CeA CRFR1 activation in adult males only, which is consistent with the age-dependent switch in CeM CRFR1-regulated changes in GABA release. However, it is important to note that the previously reported CRFR1-induced changes in GABA neurotransmission were observed in the CeM, whereas the cannula implants used in the current study allow only for specificity to the CeA as a whole. It is possible that specific activation of CeM CRFR1s would have produced a more robust behavioral change in males. Additionally, the current study tested animals during late adolescence (P45-51), a developmental stage that may fall beyond the optimal window for detecting anxiolytic effects of CeA CRFR1 activation in adolescent males.

There are several factors that could have impacted our ability to detect differences in anxiety-like behavior between groups. Importantly, we used only one behavioral task, the light-dark box, which limits our ability to draw definitive conclusions about how CeA CRFR1 activation influences anxiety-like behavior in males and females across the lifespan. There are limitations to the LDB test such as a single testing apparatus was used, which did not allow for scaling of the size of the apparatus to the size of the animals at different ages. Additionally, the LDB test does not allow for a proxy of locomotor behavior because the animal is not visible while in the dark zone. However, a recent study demonstrated that a higher dose of Stressin-1 (~4.5μM) injection into the amygdala of male Wistar rats increased anxiety-like behavior in an elevated plus maze, but did not alter generalized locomotor behavior measured by closed arm entries, suggesting that amygdala CRFR1 activation can influence anxiety-like behavior without changing locomotor behavior (Broccoli et al., 2025). In order to fully understand how CeA CRFR1 activation impacts anxiety-like behavior, a more comprehensive battery of behavioral tasks should be used. Furthermore, bilateral cannulation surgeries occurring 5-6 days prior to the LDB test could have influenced behavior during the test, as these surgeries require anesthesia and permanent cannula implantation, both of which can influence affective behaviors particularly for juveniles and adolescents (Frumberg et al., 2007; Landin et al., 2019). In particular, surgeries for the juvenile group began on P21, one day after weaning, which could have been an additional stressor that altered our ability to detect behavioral changes induced by CeA CRFR1 activation.

In the current study, we used experimental conditions designed to minimize stress, whereas chronic stress is known to activate the CEA CRF system, enhance anxiety-like behavior, and make animals sensitive to CRFR1 pharmacological manipulations. For example, following a 7-day chronic predator odor exposure, CRFR1 blockade decreased anxiety-like behavior in those exposed to predator odor, but did not have an effect on non-stressed controls tested in a basal state (Adamec et al., 2010), suggesting that predator odor stress leads to tonic activation of the CRF system through CRFR1s. In addition to chronic stress conferred by a naturalistic stressor, CRF partially regulates the negative affective state associated with withdrawal following chronic drug exposure. In general, decreasing CRF activity during drug withdrawal reduces withdrawal-induced anxiety following chronic exposure to alcohol, opiates, and nicotine (Baldwin et al., 1991; Contarino & Papaleo, 2005; Iredale et al., 2000; Marcinkiewcz et al., 2009; Patel et al., 2022; Rassnick et al., 1993). In the current study, we enhanced CRFR1 activity using one dose of a CRFR1 agonist in non-stressed animals, as an attempt to mimic the effect of a stress exposure on the CRF system and anxiety-like behavior. However, the 1 μM dose of Stressin-1 was administered to all experimental subjects based on electrophysiological evidence of age- and sex-specific effects on GABAergic transmission within the CeM (Rouzer & Diaz, 2021). We had previously demonstrated that 100 nM Stressin-1 infused into the CeA of adolescent rats also had sex specific effects on anxiety-like behavior in the LDB test, however, we chose the higher dose of 1 μM for the current study because this dose is necessary to elicit electrophysiological effects in the CeA of both adolescent and adult male and female rats, suggesting that a higher dose would be more likely to produce behavioral effects across age (Rouzer & Diaz, 2022). In line with this, a recent study showed that Stressin-1 infusion into the amgydala, targeting the basolateral amgydala-CeA junction, increased anxiety-like behavior measured in an elevated plus maze test in male Wistar rats using a higher dose (~4.5μM) than in the current study (Broccoli et al., 2025). It is possible that the in vivo dose-response relationship differs from that observed ex vivo, with sensitivity to Stressin-1 potentially influenced by age and sex.

In the present study, CRF and CRFR1 mRNA expression in the CeM and CeL did not vary across age or sex. This result is consistent with previous studies that found no basal differences in amygdala CRFR1 expression between males and females or across the lifespan in mice; as well as a report that found no differences in CeA CRFR1 binding between juvenile (P30) and adult (P98) male and female Long Evans rats (Locci et al., 2021; Weathington & Cooke, 2012). However, a study by Viau et al. reported that CRF mRNA expression doubled in the CeA between P30 and P60 in males, with a non-significant increase in females of the same age (Viau et al., 2005). One important factor influencing expression of CRF and/or CRFR1 is the prior history of the animal. We assessed CRFR1 and CRF mRNA under basal conditions, and perhaps the addition of a stressor prior to tissue collection would have revealed underlying differences in this system responding to stress across age and sex. For example, Locci and colleagues (2021) found a different pattern of age and sex effects in CRF and CRFR1 expression across the lifespan at 1, 6, 12, or 18 months in mice when assessment was done following a 2-hour restraint stress (Locci et al., 2021), with the sex and stress-induced differences becoming apparent at the 6 month time point, not as much at 1 month of age. Furthermore, exposure to early life stress increased CeA CRF gene expression, but only in females and only if the early life stress was unpredictable, suggesting that the specific type of stress paradigm may also influence the CRF system (Prusator & Greenwood-Van Meerveld, 2017). Interestingly, Viau and colleagues found that a single 30-minute restraint stress did not affect CRF mRNA expression in either sex (Viau et al., 2005). Importantly, several previous studies measured mRNA levels in the entire amygdala or the CeA as a whole whereas in the current study we used RNAscope for specific assessments of CeA subnuclei. Additionally, the lack of age and sex differences in CRF or CRFR1 mRNA does not indicate that there are no differences in CRF or CRFR1 protein levels, as the two do not necessarily directly correlate. Our assessment of the ontogeny of CRF or CRFR1 mRNA expression was limited to early periods of adolescence through early adulthood (~2.5-3 months of age). It is possible that development of the CeA CRF system may continue into later stages of adulthood (i.e. 6 months) which we did not examine. Finally, mRNA or protein expression does not fully explain functional changes, as the types of neurons CRF or CRFR1 are expressed on, the G protein-coupling mechanisms, among many other factors can contribute to the effect of CRFR1 activation on anxiety-like behavior.

Anxiety disorder symptoms are complex and can change dynamically across the lifespan differently in males and females, highlighting the need for consideration of age and sex while investigating mechanisms to target with pharmacological treatments. In the current study, we observed a sex-specific pattern of baseline anxiety-like behavior across ontogeny where adolescent males displayed higher anxiety-like behavior than adult males, suggesting that baseline anxiety decreases as males age from adolescence to adulthood. In contrast, juvenile females displayed reduced anxiety-like behavior than adult females, suggesting that baseline anxiety increases as females age from the juvenile period into adulthood. Indeed, sex differences in anxiety-related behaviors are not surprising given that females are more likely to have an anxiety disorder than males, which suggests that the neurobiological mechanisms that underlie anxiety are different between the sexes (Bangasser & Valentino, 2014; Bowman et al., 2025). Furthermore, previous reports indicate that there are sex-specific patterns of neural activation following CRF administration (Salvatore et al., 2018; Wiersielis et al., 2016). In the current study, postpubertal adolescent and adult females were tested without tracking the estrous cycle. Importantly, intracerebroventricular CRF-induced increases in anxiety-like behavior are influenced by the estrous cycle stage in rats (Wiersielis et al., 2016), demonstrating that CRF manipulations may produce different behavioral outcomes in females depending on their current estrous phase. Specifically within the CeA, there is a population of CRF neurons that are colocalized with androgen and estrogen receptors (Rybka et al., 2023), representing a potential mechanism through which gonadal hormones could influence function of CeA CRF neurons. However, we found that CeA CRFR1 activation had an anxiolytic effect in females, suggesting that CRF may promote anxiety-like behavior in females through a mechanism outside of the CeA. Clearly, more studies are needed to fully understand the intricacies of the CRF system in regulating anxiety.

The CRF system has long been an intriguing target for anxiety disorder treatment development. However, evidence from clinical trials evaluating the efficacy of CRFR1 antagonists as anxiety disorder treatments have provided mixed results, demonstrating the need for a more careful examination of the precise neural mechanisms through which the CRF system promotes anxiety-like behavior (Coric et al., 2010; Dunlop et al., 2017; Ising et al., 2007; Kwako et al., 2015; Lee et al., 2023; Morabbi et al., 2018; Schwandt et al., 2016). The current study suggests that the usefulness of CRFR1 antagonists as treatments for anxiety disorders likely varies due to a combination of factors, including age and sex. Furthermore, previous work from our lab and others suggests that early-life insults may alter the developmental trajectory of the immature CRF system, ultimately leading to long-lasting changes in stress responsivity. For example, a single moderate prenatal alcohol exposure during the 12th gestational day in rats blunted the effects of Stressin-1 on neurons of the CeA in adolescent offspring (Rouzer & Diaz, 2022), suggesting that targeting this receptor as a treatment may not work as well in this population. It is possible that other insults during prenatal or postnatal development, such as maternal deprivation or early life adversity, may alter the function of the CRF system in a chronic manner that makes this method of treatment differentially efficacious (Brunton et al., 2011; Demaestri et al., 2024; Dixon et al., 2025; Short et al., 2023; Vazquez et al., 2006). Taken together, the current study highlights the critical need to consider different factors including developmental history that can affect functioning of the CeA CRF system. In conclusion, this study provides further evidence that the CeA CRFR1 system contributes to anxiety-like behavior in age- and sex-specific manner, underscoring the importance of considering the complexity of this system when translating preclinical findings to the clinical setting.

Supplementary Material

Supplementary figures

Supplementary Figure 1. Representation of location of drug infusion into the CeA. The entire CeA is outlined in dotted blue lines. The general area of drug infusion is shown in by the filled in blue shape. Images are adapted from Swanson, L.W. (2004) Brain maps: structure of the rat brain, 3rd edition.

Supplementary Figure 2. There were no differences in CRFR1 or CRF total transcript number in the CeM (B, D) or the CeL (A, C).

Acknowledgements

This study was funded by NIAAA grants R01AA028566, F32AA031185, P50AA017823, and T32AA025606.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary figures

Supplementary Figure 1. Representation of location of drug infusion into the CeA. The entire CeA is outlined in dotted blue lines. The general area of drug infusion is shown in by the filled in blue shape. Images are adapted from Swanson, L.W. (2004) Brain maps: structure of the rat brain, 3rd edition.

Supplementary Figure 2. There were no differences in CRFR1 or CRF total transcript number in the CeM (B, D) or the CeL (A, C).

NIHMS2162132-supplement-Supplementary_figures.pptm (1.6MB, pptm)

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