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. Author manuscript; available in PMC: 2026 Feb 1.
Published in final edited form as: Pharmacol Biochem Behav. 2024 Dec 1;247:173931. doi: 10.1016/j.pbb.2024.173931

Region-specific neuroadaptations of CRF1 and CRF2 expression following heroin exposure in female rats

Piper Schneider 1,*, Danielle Goldbaum 1,*, Ansh Agarwal 3, Ashton Taylor 3, Peyton Sundberg 1, Eliot L Gardner 3, Robert Ranaldi 2,4, Zhi-Bing You 3, Ewa Galaj 1
PMCID: PMC11769769  NIHMSID: NIHMS2039760  PMID: 39626795

Abstract

While stress increases vulnerability to development of addiction, the recruitment of corticotropin releasing factor (CRF) with excessive drug use heightens the risk of stress-induced relapse. CRF signaling is transmitted via CRF1 and CRF2 receptors, but the roles of these receptors in heroin self-administration and related neuroadaptations of the CRF system within mesolimbic brain loci are not well understood. In this study, we first investigated the causal role of CRF1 and CRF2 receptors in heroin self-administration. Intracerebroventricular (ICV) microinjections of antalarmin (a CRF1 antagonist) or astressin-2B (a CRF2 antagonist) caused brief, dose-dependent reductions in heroin self-administration in female rats, suggesting that these receptors play a critical role in heroin-motivated behaviors. We then used western blotting to examine neuroadaptive changes to CRF1 and CRF2 receptor expression in key forebrain and midbrain regions associated with opioid addiction. Female Long Evans rats treated with escalating doses of heroin for 16 days demonstrated significantly higher naloxone-precipitated withdrawal symptoms than saline-treated rats. Heroin-treated rats showed a significant decrease in CRF1 receptor protein expression in the ventral tegmental area (VTA) and an increase in the nucleus accumbens (NAc) but no changes in the prefrontal cortex (PFC), insula, dorsal striatum (dSTR), dorsal hippocampus (dHippo), anterior hypothalamus (HYPTH), amygdala, or substantia nigra (SN) as compared to saline-treated rats. After chronic heroin exposure, CRF2 receptor expression was significantly downregulated in the dHippo, VTA and HYPTH but not in the other brain regions we investigated. The results of this study suggest that: (1) CRF1 and CRF2 receptors play an important role in self-administration and (2) heroin exposure may lead to region-specific neuroadaptation of CRF1 and CRF2 receptors. Such neuroadaptations might in part contribute to the continuation of drug use and stress-induced relapse.

Keywords: CRF1, CRF2, heroin, drug self-administration, nucleus accumbens, neuroadaptations, withdrawal symptoms, naloxone, ventral tegmental area, opioid

1. Introduction:

While stress is a well-known risk factor for the development of opioid addiction and a strong predictor of relapse, continued drug use leads to potentiation of stress-like mechanisms, including a recruitment of corticotropin releasing factor (CRF) signaling via CRF1 and CRF2 receptors (Armario, 2010; Baidoo and Leri, 2022; Contarino and Papaleo, 2005; Koob, 2010). Several studies localized CRF1 and CRF2 receptors in key forebrain regions involved in opioid abuse and addiction, including various hypothalamic regions, the frontal cortex, insula, hippocampus (Hippo), amygdala, nucleus accumbens (NAc) and striatum (Chalmers et al., 1995; Primus et al., 1997; Sánchez et al., 1999). CRF1 and CRF2 receptors have been also identified in midbrain regions such as the substantia nigra (SN) and ventral tegmental area (VTA) (Chen et al., 2000; Sharpe et al., 2022).

Although much attention has been given to the involvement of CRF1 receptors in addiction, the specific role of CRF2 receptors in opioid abuse has yet to be elucidated. Previous studies have demonstrated a link between CRF1 receptors and mechanical hypersensitivity to pain in heroin-treated rats (Edwards et al., 2012; Park et al., 2015). Studies have also established a causal role of these receptors in stress-induced reinstatement of drug-seeking behavior (Shaham et al., 1998; Wang et al., 2007). CRF1 antagonists block the acquisition of opiate withdrawal-induced conditioned place aversion (Stinus et al., 2005) and naloxone-precipitated withdrawal signs in rodents (Almela et al., 2012; Navarro-Zaragoza et al., 2014). We and others have previously reported that local blockade of CRF1 receptors in the VTA reduces heroin self-administration in male rats (Galaj et al., 2023) and that CRF1 antagonism attenuates the escalation of heroin intake with long-access to the drug (Park et al., 2015), suggesting that these receptors play an important role in opioid abuse. Likewise, CRF2 antagonism attenuates stress-induced cocaine seeking (Wang et al., 2007) and reduces naloxone-precipitated somatic signs of opiate withdrawal in rats (Navarro-Zaragoza et al., 2011; Skelton et al., 2007). Research with transgenic CRF1 or CRF2 receptor knockout mice (CRF1 −/− and CRF2 −/−) has demonstrated that opioid withdrawal symptoms are prolonged in the absence of CRF1 receptors and shortened in the absence of CRF2 receptors, suggesting that CRF1 and CRF2 receptors play differential roles in the stress response and in drug withdrawal (Morisot and Contarino, 2016).

While several studies report neuroadaptations of CRF and CRF receptors following drug and alcohol exposure, there is still limited knowledge regarding how repeated heroin exposure impacts protein expression of CRF1 and CRF2 receptors within the mesolimbic regions, especially in females. A significant increase in CRF mRNA expression has been observed in the amygdala and paraventricular nucleus (PVN) of the hypothalamus (HYPTH) in response to morphine dependence (Navarro-Zaragoza et al., 2011). We have previously reported that heroin self-administration in male rats leads to an upregulation of CRF1 mRNA and protein expression in the VTA (Galaj et al., 2023). When exposed to a stressor such as a forced swim test, alcohol intake increases in alcohol-treated rats, which correlates with an upregulation of CRF mRNA expression in the central amygdala and CRF1 receptor mRNA expression in both the medial and basolateral (BLA) amygdala. In contrast, CRF2 receptor mRNA expression decreases in the BLA in response to stress in alcohol-treated rats (Sommer et al., 2008). Repeated cocaine or methamphetamine treatment also produces bi-directional alterations of CRF2 receptors in midbrain dopamine neurons versus astrocytes (Sharpe et al., 2022). However, to date, there has been no systematic study comparing/contrasting heroin-induced neuroadaptations of CRF1 and CRF2 receptors across different midbrain and forebrain regions.

Furthermore, it is well known that females are more susceptible to stress and twice as likely to develop stress-related disorders than males (Remes et al., 2016, Steel et al., 2014). While female rodents show more robust changes in CRF and CRF1 expression across the lifespan than males (Locci et al., 2021) and have higher hypothalamic-pituitary-adrenal (HPA) axis reactivity than males (Goel et al., 2014; Weinstock et al., 1992), the impact of heroin exposure-related stress on the female brain is less well understood. This is in part because decades of research have routinely excluded female subjects due to potential large variability in the data (Fields, 2014) [but see (Becker et al., 2016)]. Thus, given higher susceptibility to stress-related disorders and our limited understanding of female brain/behavior pathology, the present study focused on understanding the impact of an escalating regimen of heroin exposure on CRF1 and CRF2 protein expression in female rats. We were particularly interested in investigating potential changes in CRF1 and CRF2 receptor expression in the prefrontal cortex (PFC), insula, NAc, dorsal striatum, HYPTH, dHippo, amygdala, VTA, and substantia nigra (SN) as these regions are known to play a critical role in opioid-motivated behaviors. In addition, this study investigated the involvement of these receptors in intravenous heroin self-administration (IVSA).

2. Methods:

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 2011) and was approved by the Colgate University Institutional Animal Care and Use Committee as well as the National Institute on Drug Abuse Institutional Animal Care and Use Committee. This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

2.1. Animals:

The subjects were female Long Evans rats (n=68) taken from our in-house colonies from breeders purchased from Envigo (Amsterdam, NY). Rats were housed together on a reversed 12 h light:12 h dark cycle (lights on at 7 pm) in temperature- (23 °C) and humidity- (40–60%) controlled rooms. All rats had access to food (LabDiet chow) and water at all times.

2.2. Surgeries:

Each rat was anesthetized with ketamine/xylazine (90/10 mg/kg, i.p.). The scalp and the neck of the rat were shaved, and cleaned with iodine, and an ophthalmic ointment (Paralube Vet ointment) was applied to the eyes to prevent corneal drying. During and after surgery the rats were placed on heating pads to prevent hypothermia. For catherization, as described in our previous studies (Galaj et al., 2015, 2014), a small incision was made to the right of the midline of the neck. The jugular vein was exposed and the tip of a silastic catheter (Dow Corning, Midland, MI) was inserted into the right atrium of the heart. The catheter was secured to the vein with silk sutures and its free end was fed subcutaneously around the back of the neck to exit through the scalp incision. Next, the catheter was connected to a bent 22-gauge stainless steel connector. Two holes were drilled through the skull above the lateral ventricles and two stainless-steel guide cannulae (23 gauge/16 mm long) were implanted bilaterally into the lateral ventricles using the following coordinates: AP −0.8, ML ±2.1, and DV −4.3 at 10° angle away from the midline (Paxison and Watson, 2013). The cannulae and catheter connector were permanently fixed to the skull using Gorilla superglue and dental acrylic anchored to four stainless steel screws screwed into the skull. Obturators were inserted into the cannulae to prevent blockage and remained there at all times except during microinjections. After surgery, the rats were placed in a warm incubator and were closely monitored during post-surgery recovery and experimentation periods. To maintain its patency, the catheter was filled with 0.05 ml of heparin (30 U/ml of saline) and gentamicin (1 mg/ml) immediately after surgery and daily thereafter. The rats were given five days of recovery before IVSA began.

2.3. Drugs:

Heroin for the withdrawal experiment was obtained from the NIDA Drug Supply Program and was dissolved in saline to achieve 2, 3, 4, 5 and 6 mg/ml concentration. Heroin for the IVSA study was obtained from the NIDA IRP Pharmacy and dissolved in saline to achieve a 0.05 mg/kg/inj dose. Antalarmin, a CRF1 receptor antagonist (Chen et al., 1997) was purchased from Tocris Bio-Techne (Minneapolis, MN) and dissolved in a vehicle solution consisting of 10% DMSO and 15% Tween 80. Astressin-2B, a CRF2 antagonist (Hoare et al., 2005; Rivier et al., 2002) was purchased from Tocris Bio-Techne and dissolved in saline. Doses of antalarmin and astressin-2B were selected based on previous studies involving microinjections (Galaj et al., 2023; Gondré-Lewis et al., 2016; Walker et al., 2017)

2.3. Operant conditioning chambers:

Heroin self-administration took place in standard Med-Associates operant conditioning chambers placed in a ventilated, sound-attenuating cubicle with an operating fan. Each chamber was equipped with two retractable levers, a white light above each lever and a drug line consisting of a metal tether covering a polyethylene tubing which, through a fluid swivel, was connected to a syringe pump (Razel, 3.33 rpm) loaded with a 10 ml syringe.

2.4. Procedures:

2.4.1. Assessing the impact of intra-cerebroventricular (ICV) antalarmin or astressin-2B on heroin self-administration:

Female Long Evans rats were trained to self-administer heroin under a fixed ratio 1 (FR1) schedule of reinforcement during daily 3-h sessions. Presses on the active lever activated the white cue light above the active lever and the pump delivering heroin solution (0.05 mg/kg/injection) over 4.5 s. Presses on the inactive lever were counted but had no consequences. Rats continued IVSA training for 7–10 days until reaching a criterion of stable responding (defined as 3 consecutive sessions in which the total number of infusions taken per session did not vary by more than ±10% of the mean of the three sessions). Next, rats were divided into 6 groups and were tested with intracerebro-ventricular (ICV) antalarmin (a CRF1 antagonist; Exp.1A) or astressin-2B (a CRF2 antagonist; Exp.1B) at different doses. Prior to the test sessions, obturators were removed and microinjectors, extending 1 mm beyond the guide cannulae, were inserted into the cannulae. Bilateral microinjections of antalarmin [0 (n=7), 4 (n=7) or 8 (n=9) μg/μl/side] or astressin-2B [0 (n=7), 4 (n=10) or 8 (n=8) μg/μl/side] were delivered into the lateral ventricle over 60 s, using a 10 μl Hamilton syringe and the pump. Microinjectors were kept in place for an additional 2 min to allow the drug to diffuse. Next, the obturators were inserted back into the guide cannulae and the rats were placed into the operant conditioning chambers where they self-administered heroin, as previously.

2.4.2. Assessing the impact of chronic heroin treatment on expression of CRF1 and CRF2 receptors in various brain regions:

Different groups of rats were used in this experiment. We used a heroin exposure protocol adapted from Marchette et al (Marchette et al., 2021). Rats were injected with 1 ml/kg subcutaneous (sc) saline or heroin at escalating dose regimen (2–6 mg/kg; sc) for 16 days. The initial dose of heroin was 2 mg/kg and it was increased by 1 increment each day (2, 3, 4, 5, 6 mg/kg) until reaching a maximum dosage of 6 mg/kg (Day 5). This dose was then administered to the animals until the last day of the treatment (Days 5–16). On the test day (Day 16), 3 hours after the last treatment injections, rats were injected subcutaneously with 3 mg/kg of naloxone (a μ opioid antagonist) and withdrawal symptoms were recorded within 10 minutes of the administration. Weights were recorded twice: immediately before the naloxone injection and 1 hour after the naloxone treatment.

We then performed histology to verify cannula placement. Five rats were eliminated from the study.

2.4.3. Blind behavioral scoring:

Rats were videotaped for 30 minutes following naloxone administration and scored by 2 experimenters blind to subject-group assignment, who used a scoring system adapted from (Marchette et al., 2021). Withdrawal symptoms such as teeth chattering, jumps and wet dog shakes were scored as 1–3 depending on the number of occurrences, but urination as 1, defecation or diarrhea as 2, genital grooming, vocalization and abnormal posture as 3 and profuse salivation as 7, regardless of the number of occurrences (see Fig. 3). Each gram of weight loss within an hour post-naloxone treatment was scored as 1. The final withdrawal score was then obtained as a sum of all scores. The final scores obtained by each of the experimenters were averaged and used to compare the saline (n=10) and heroin-treated (n=10) groups.

Fig. 3A-B.

Fig. 3A-B.

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Naloxone-precipitated withdrawal symptoms in female rats. (A) shows the behavioral measures used for scoring. (B) Naloxone (3 mg/kg; sc) induced significantly higher withdrawal symptoms in heroin- than saline-treated rats (*p<0.05).

2.4.4. Western Blot assay:

Following naloxone-precipitated withdrawal, on Day 17, rat brains were extracted and rapidly submerged in cold methyl-butane. Brain sections were harvested using the cryostat and scalpel to dissect desired brain regions. Samples were frozen and later homogenized with 1X RIPA lysis buffer followed by centrifugation at 14,000 rpm at room temperature for 10 min. Total protein concentrations in each sample were then determined using a Pierce BCA protein standard Kit and samples were diluted to a final concentration of 2 μg/μl. Right before gel electrophoresis, samples were denatured by rapid heating for 5 minutes at 95°C and cooling. The NuPage Mini Protein Gel (Bis-Tris 4–12%; Fisher) was then loaded with the 15 μl of each sample and 10 μl of protein ladder (Bio-Rad Precision Plus Protein Dual Color Standard) and run for 1.5 hours at 150V. Next, the gel was removed and transferred onto a PVDF membrane (Millipore Immobilon; 0.45 μm), followed by air drying of the membrane for 1 hour. The membrane was then rehydrated for 10 seconds in methanol and rinsed in distilled water twice. The membrane was then washed in phosphate buffer saline (PBS) 3 times for 5 minutes each and incubated in 5% nonfat milk/PBS1X blocking solution for 2 hours. The membrane was then incubated overnight in primary goat anti-CRF1 antibodies, (Novus biological, cat # NBP100175; 1:1,000 dilution), and mouse anti-beta-actin (Cell signaling technology; cat # 8H10D10; 1:2,000 dilution) diluted in the 5% nonfat milk/PBS1X blocking solution. The following day, the membrane was washed 3 times for 5 minutes each in PBST and 1X PBS and incubated in secondary donkey anti-goat antibodies (Abcam; cat #AB97110) and donkey anti-mouse (Abcam, cat #AB205724) both at a 1:10,000 dilution, for 2 hours. The membrane was then washed 3 times for 5 minutes each in PBST and 1 x in PBS. It was then incubated in SuperSignal West Pico Plus chemiluminescent substrate (ThermoFisher Scientific, cat # 34580) for 5 minutes before scanning. The membrane was then stripped with Western Blot stripping buffer (15 min) and rinsed 3 times for 5 minutes each in PBS. Blocking solution was reapplied for 1 hour before the membrane was incubated in CRF2 primary rabbit anti-CRF2 antibodies (Abcam, cat # ab104368; 1:1000) and mouse anti-beta-actin antibodies (1:2,000), overnight. The next day, the membrane was washed 3 times for 5 minutes each in PBST followed by one wash in PBS and incubation in secondary CRF2 antibodies, donkey anti-rabbit (1:10,000) and donkey anti mouse (1:10,000), for 2 hours. The membrane was washed 3 times for 5 minutes each in PBST followed by one wash in PBS and incubated for 5 minutes in chemiluminescent substrate before scanning. Immediately after, individual blots were imaged with a Bio-Rad ChemiDoc XRS+ Scanner.

2.5. Data analysis:

In Exp.1, the number of infusions during baseline sessions and test sessions with antalarmin or astressin-2B were analyzed per 3 hours using a separate two-way (dose x phase) ANOVA. Heroin infusions as percent baseline were analyzed per one-hour-bin, using separate one-way ANOVAs. Significant effects were followed by Tukey’s post hoc tests. Behavioral data from Exp.2 was analyzed by converting heroin withdrawal raw scores into defined values as outlined above, and comparing saline vs heroin groups using an independent t-test. Scoring between blind observers was also compared to determine inter-observer reliability via Cronbach’s alpha. Western blot data was analyzed using Image Lab software to generate volumetric ratios of the target proteins (CRF1 or CRF2) in relation to the control of β-actin for each run. The adjusted volume ratios were compared between saline and heroin rats for each brain region and each protein within each brain region using independent t tests, for specific region.

3. Results

3.1. Intracerebroventricular (ICV) injections of antalarmin significantly reduced heroin IVSA in female rats.

In this experiment, we investigated an involvement of CRF1 receptors in heroin IVSA. During baseline, female rats in all groups self-administered on average 22 heroin infusions per session (0.05 mg/kg/inf). ICV-antalarmin produced no significant changes in heroin intake within 3-hour sessions. This observation was confirmed by a two-way ANOVA revealing no main effect of dose (F2, 20=1.35; p=0.279), phase (F1,20=0.74; p=0.39) nor dose x phase interaction (F2,20=2.49; p=0.108). However, a close analysis of the infusion data per 1-hour bin as percentage baseline, revealed that ICV-antalarmin produced an attenuating effect in the first hour of heroin IVSA (Fig. 1A) but not in the second or third hour (Fig. 1B and Fig. 1C). Rats microinjected with 8 μg/μl of antalarmin self-administered significantly less heroin than vehicle-treated rats in the first hour. A one-way ANOVA on percent baseline data revealed a significant dose effect (F2,20=7.65; p=0.0034). Tukey’s post hoc test revealed a significant difference between the 8 μg/μl of dose and vehicle. No significant group differences were observed in the second (F2,20=1.014; p=0.3808; Fig. 1B) or third hour (F2,20=1.006; p=0.38; Fig. 1C). This suggest that antalarmin produced significant but short-lasting effects. There was no significant effect of antalarmin on active and inactive lever presses/3 hours (F2,20=1.98; p=0.163 and F2,20=1.95; p=0.168, respectively; Fig. 1D and Fig. 1E).

Fig. 1A-E.

Fig. 1A-E.

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The impact of intracranial antalarmin (a CRF1 antagonist) on heroin IVSA in female Long Evans rats. (A) Intracerebroventricular (ICV) microinjections of antalarmin at the 8 μg/μl/side dose produced significant reductions in percent baseline heroin infusions in the first hour of IVSA as compared to vehicle (*p<0.05) (B-C) No changes in heroin intake were observed in the second (B) and third hour (C) with the antalarmin treatment (p>0.05, as compared to vehicle). No significant changes in active (D) or inactive (E) lever presses were observed when analyzed a total number of responses within 3 hours.

3.2. Intracerebroventricular (ICV) injections of astressin-2B significantly reduced heroin IVSA in female rats.

Next, we investigated the causal role of CRF2 receptors in heroin IVSA. Rats showed a similar pattern of heroin IVSA during baseline and test sessions, regardless of the treatment. A two-way ANOVA on the total heroin infusions revealed no significant main effect of dose (F2,22=01.65; p=0.214), phase (F1,22=0.016; p=0.89) nor dose x phase interaction (F2,22=1.87; p=0.177). However, a careful analysis of the infusion data per every hour as percent baseline revealed that the highest dose of astressin-2B caused a significant reduction in heroin intake in the first hour (F2,22=4.102; p=0.030; Fig. 2A). No changes in heroin IVSA were detected in the second- (F2,22=0.23; p=0.79; Fig. 2B) or third-hour post-astressin-2B treatment (F2,22=0.36; p=0.70; Fig. 2C). There was no significant effect of astressin-2B on active and inactive lever presses/3 hours (F2,22=2.711; p=0.087 and F2,22=3.08; p=0.065, respectively; Fig. 2D and Fig. 2E).This suggests that Astressin-2B produced significant but short-lasting effects on heroin IVSA.

Fig. 2A-E.

Fig. 2A-E.

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The impact of intracranial astressin-2B (a CRF2 antagonist) on heroin IVSA in female Long Evans rats. (A) Intracerebroventricular (ICV) microinjections of astressin-2B at the 8 μg/μl/side dose produced a significant reduction in percent baseline heroin infusions in the first hour of IVSA as compared to vehicle (*p<0.05). (B-C) No changes in percent baseline heroin intake were observed in the second (B) or third hour (C) with the astressin-2B treatment (p>0.05 as compared to vehicle). No significant changes in active (D) or inactive (E) lever presses were observed when analyzed a total number of responses within 3 hours.

3.3. Escalating regimen of heroin exposure leads to region-specific changes in CRF1 and CRF2 protein expression.

The purpose of the present study was to elucidate neuroplastic changes in CRF1 and CRF2 receptor expression in brain regions implicated in opioid abuse. Fig. 3B shows behavioral scoring data generated by 2 blind observers (ɑ=.72) based on naloxone-precipitated withdrawal signs outlined in Fig. 3A. An escalating regimen of heroin treatment (2–6 mg/kg/sc.) for 16 days led to more severe withdrawal (and higher score) in heroin than saline-treated rats during the naloxone-precipitated withdrawal test (t18=5.40, p<0.001).

In addition to behavioral data comparisons, CRF1 receptor expression in the VTA was reduced in heroin rats as compared to saline rats (t18=2.42, p=0.02; Cohen d=1.08; Fig. 4A). In contrast, CRF1 receptor expression in the NAc was greater in heroin-treated rats (t18=2.30, p=0.03; Cohen d= −1.03; Fig. 4B). No significant differences in CRF1 receptor expression were detected in the PFC (t18=0.52, p=0.60; Fig. 4C), insula (t18=0.39, p=0.69; Fig. 4D), striatum (t18=0.01, p=0.98; Fig. 4E), HYPTH (t18=0.14, p=0.88; Fig. 4F), dHippo (t18=0.51, p=0.61; Fig. 4G), amygdala (t18=0.26, p=0.79; Fig. 4H) or SN (t18=0.05, p=0.95; Fig. 4I) between saline- and heroin-treated groups.

Fig. 4A-I.

Fig. 4A-I.

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CRF1 protein expression in key brain regions after saline (S) or heroin (H) treatment. (A) After heroin exposure protein expression of CRF1 receptors significantly decreased in the VTA and (B) increased in the NAc (*p<0.05 as compared to saline group). No significant changes in CRF1 expression were observed in (C) the prefrontal cortex, PFC, (D) insula, (E) hypothalamus, HYPTH, (F) dorsal hippocampus, dHippo, (G) amygdala, AMY or (I) substantia nigra, SN (p>0.05 as compared to saline group.

In this study, we also found that CRF2 protein expression was significantly decreased in the VTA (t18=1.70, p=0.05; one-tailed; Cohen d=0.76; Fig. 5A), HYPTH (t18=1.78, p=0.04; one-tailed; Cohen d=0.78; Fig. 5B) and dHippo (t18=2.28, p=0.03; Cohen d=1.02; Fig. 5C) in heroin-treated rats. Similar levels of CRF2 protein expression were observed in the heroin and saline rat PFC (t18=0.38, p=0.70; Fig. 5D); insula (t18=0.33, p=0.73; Fig. 5E), NAc (t18=0.51, p=0.61; Fig. 5F), striatum (t18=0.47, p=0.64; Fig. 5G), amygdala (t18=0.12, p=0.90; Fig. 5H), or SN (t18=0.45, p=0.65; Fig. 5I). Thus, our results indeed indicate region-specific changes in CRF1 and CRF2 receptor expression in females following an escalating regimen of heroin exposure.

Fig. 5A-I.

Fig. 5A-I.

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CRF2 protein expression in key brain regions after saline (S) or heroin (H) treatment. (A) After heroin exposure, protein expression of CRF2 receptors significantly decreased in the VTA, (B) HYPTH, and (C) dHippo (*p<0.05 as compared to saline group). No significant changes in CRF1 expression were observed in (D) the PFC, (E) insula, (F) NAc, (G) dorsal striatum (H) amygdala or (I) SN (p>0.05 as compared to saline group).

4. Discussion:

There is an interesting intersection between the mesolimbic system and the CRF system. Opiates potently activate the mesolimbic “reward” pathway, and their repeated use leads to dependence that hijacks the CRF system (Koob, 2010). Concurrently, hypersensitivity of the CRF system increases the stress response, thereby increasing the propensity to relapse (Heinrichs et al., 1995). We hypothesized that heroin exposure at escalating doses is associated with neuroadaptations of CRF1 and CRF2 receptors, which play important roles in heroin self-administration as well as in the stress response.

In the present study, we found that ICV-microinjections of antalarmin or astressin-2B at the highest dose (8 μg/μl) significantly reduced heroin IVSA but these effects were short-lasting. The effects of antalarmin and astressin-2B administration were evident in the first hour of heroin IVSA. The short-lasting effects of antalarmin most likely are related to its half-life (half-life=1.5 hours with intravenous self-administration and half-life=0.5–1 hour with oral administration) in rats (Chen et al., 1997). The effects of astressin-2B were previously reported to be still evident 45 min post-injection (Rivier et al., 2003; Wang et al., 2013). Overall, these findings suggest that both CRF receptors play a role in heroin IVSA. Antalarmin- or astressin-2B-induced reductions in self-administered heroin infusions cannot be explained by general reduction in motoric impairment as neither of the drugs affected inactive lever responding. We also reported previously that systemic injections of antalarmin do not affect locomotion in rats (Galaj et al., 2023). Others reported that intraventricular injections of astressin-2B do not affect locomotor activity in rats (Buzas et al. 2019).

While it is well established that the hypothalamic-pituitary-adrenal (HPA) axis plays a strong role in addiction and relapse (Burke and Miczek, 2014; Koob, 2015; Zorrilla et al., 2014), evidence showing specific changes in this system, especially in female rats, remains sparse. In this study, we found that 16 days of heroin treatment (with escalating doses) induced physical withdrawal in female rats. In contrast to saline-treated rats, heroin-treated rats showed more withdrawal signs including rapid weight lost, wet dog shakes, diarrhea, and abnormal posture. We also found that CRF1 receptor protein expression was decreased in the VTA but increased in the NAc after prolonged heroin exposure. No significant changes in CRF1 expression were detected in the PFC, insula, dorsal striatum, amygdala, dHippo, HYPTH or SN. While past studies have focused on CRF1 receptors more extensively than CRF2 receptors (Galaj et al., 2023; Greenwell et al., 2009; Park et al., 2015), a growing body of evidence suggests that CRF2 receptors may be an important site for neuroplastic changes in chronic drug use. In this study, we found significant downregulation of CRF2 receptors in the VTA, HYPTH, dHippo in rats exposed to heroin treatment. No changes in CRF2 receptors were detected in the PFC, insula, NAc, dorsal striatum, amygdala or SN.

4.1. The role of CRF1 receptors in opioid-driven behaviors

Previous studies have long established the role of CRF1 receptors in stress- (Kehne and Cain, 2010) and opioid-related behaviors (Koob, 2010; Park et al., 2015). CRF1 receptor antagonism is known to reduce heroin intake (Galaj et al., 2023; Greenwell et al., 2009; Park et al., 2015) and stress-induced opioid seeking (Shaham et al., 1998). Antalarmin can block the acquisition and expression of conditioned place aversion produced by precipitated opiate withdrawal as well as diminish severity of withdrawal signs (Almela et al., 2012; Iredale et al., 2000; Navarro-Zaragoza et al., 2014; Stinus et al., 2005). In addition, previous studies have demonstrated the involvement of CRF1 receptors in the mechanical hypersensitivity to pain that occurs after chronic opiate exposure (Edwards et al., 2012; Park et al., 2015). These findings parallel our present results demonstrating a causal role of CRF1 receptors in heroin IVSA and neuroadaptations of CRF1 receptors in the brain’s mesolimbic system, namely the VTA and NAc.

The VTA is a major output of hypothalamic CRF-containing neurons, where CRF modulates excitatory neurotransmission (Borgland et al., 2010) and, along with CRF1 receptors, has been implicated in stress-induced drug seeking (Blacktop et al., 2011; Chen et al., 2014; Vranjkovic et al., 2018; Wang et al., 2007). As opiate use enhances CRF signaling (Armario, 2010; Koob, 2010), VTA neurons expressing CRF1 receptors (mainly dopaminergic neurons) (Galaj et al., 2023; Zhu et al., 2023) are vulnerable to excessive stimulation. As our data suggest, CRF1 receptors in the VTA become downregulated following heroin exposure, most likely as a compensatory response to excessive CRF signaling. We previously reported that VTA CRF1 mRNA and protein expression are upregulated in rats self-administrating heroin and can be reversed by environmental enrichment (Galaj et al., 2023). This discrepancy in our results most likely derives from the fact that CRF1 expression is dynamic and depends on the treatment type as well as severity of stress. For example, a previous study reported that rats implanted with morphine pellets for 5 days and experiencing naloxone-precipitated withdrawal showed significant downregulation of CRF1 mRNA in the dorsal striatum and NAc. However, those subjected to chronic morphine treatment but not withdrawal showed no changes in CRF1 mRNA in these regions (Iredale et al., 2000). These and present findings suggest that the recruitment of CRF and related neuroadaptation of CRF1 receptors might depend on the severity of stress. However, there is also the possibility that CRF1 receptor expression is regulated differentially by passive versus active drug administration. There is evidence that response contingent compared to non-contingent cocaine administration differentially affects plasma CRF in monkeys (Broadbear et al., 1999). In the present study, animals received experimenter-administered subcutaneous injections of heroin while in our previous study, animals self-administered heroin voluntarily (Galaj et al., 2023). Thus, it is conceivable that bidirectional neuroadaptation of CRF1 receptors in the VTA depends on the severity of stress and/ or contingency of drug administration. As this is beyond of the scope of the present study, our future studies will seek to address this idea.

Furthermore, we found that the expression of CRF1 receptors in the NAc was significantly increased after heroin treatment, which parallels our recent findings demonstrating that heroin IVSA leads to upregulation of CRF1 receptors in the NAc, a brain region heavily implicated in reward and motivation (Galaj et al., 2023). Although the CRF system within the limbic structures is uniquely positioned to mediate stress-induced relapse, there is evidence suggesting that activation of CRF-expressing neurons in the NAc can also magnify incentive motivation for sucrose or cocaine reward in the absence of distress (Baumgartner et al., 2022). Overexpression of accumbal CRF can increase nicotine IVSA and food intake in female rats, leading to larger CRF1 and CRF2 gene expression (Uribe et al., 2020). Local injections of a CRF1 antagonist into the NAc or amygdala can attenuate morphine-primed reinstatement of morphine conditioned place preference but have no effect on stress-induced reinstatement of conditioned place preference via foot shock (Wang et al., 2006). Thus, these data suggest that CRF in the NAc might reinforce drug taking and seeking, in the absence of distress.

4.2. The role of CRF2 receptors in opioid-driven behaviors

While CRF2 receptors have lower affinity for CRF than CRF1 receptors and have been less researched, some studies suggest that these receptors play an important role in attenuating stress mechanisms and promoting drug seeking. Pharmacological blockade (Navarro-Zaragoza et al., 2011; Skelton et al., 2007) or genetic deletion of CRF2 receptors (Ingallinesi et al., 2012; Morisot and Contarino, 2016; Papaleo et al., 2008) eliminate opioid induced withdrawal distress and minimize somatic withdrawal symptoms in rats and mice. Stimulation of CRF2 receptors, particularly in the VTA, appears to facilitate glutamate release in the NAc, which in turn prompts drug seeking. Indeed, intra-VTA CRF2 antagonism attenuates stress-induced cocaine seeking (Wang et al., 2007). While complete deletion of CRF2 receptors does not impair motivation to work for food in drug-naïve mice, it reduces the increases in motivation for palatable food in CRF2−/− mice with a history of morphine treatment (Morisot et al., 2015; Rouibi and Contarino, 2013). These findings suggest that CRF2 receptors might play a unique role in motivational effects of opiate withdrawal.

In the present study, we demonstrated that ICV-microinjections of astressin-2B attenuate heroin IVSA in female rats, providing evidence that CRF2 receptors play an important role in drug taking/seeking. We also found that the expression of CRF2 receptors in the VTA, HYPTH and dHippo significantly decreased with the escalating regimen of heroin exposure. This may be in part due to excessive release of CRF in these regions. Our results are in concert with previous findings demonstrating that maternal separation as a stressor decreases CRF2 expression in the HYPTH (Eghbal-Ahmadi et al., 1997), Hippo, amygdala and dorsal raphe nucleus (Bravo et al., 2011). Likewise, Wistar-Kyoto rats, genetically stress-sensitive rats, show higher CRF1 mRNA expression in the Hippo, dorsal raphe and amygdala but lower CRF2 mRNA expression in the dorsal raphe (Bravo et al., 2011). In contrast, higher levels of CRF2 mRNA have been reported in the PFC and Hippo after nicotine sensitization in rats (Carboni et al., 2018).

While we observed downregulation of CRF2 receptors in the VTA, HYPTH and dHippo of heroin-treated rats, we found no significant changes in CRF2 receptor expression in the PFC, insula, NAc, dorsal striatum, amygdala, or SN. This suggests that prolonged heroin exposure causes region-specific neuroadaptive changes and other, non-affected, regions are either more stress-resilient or play a less important role in opioid dependence. These findings aligned with a previous study reporting no significant changes in CRF2 mRNA in the parietal cortex, lateral septum or amygdala in morphine-treated rats (Iredale et al., 2000). To our knowledge, no studies have demonstrated a causal role of the SN in opioid dependence, despite the fact that SN GABA neurons have been implicated in heroin self-administration and relapse (Galaj et al., 2020). Of important note, the role of CRF2 expression in the insula, dorsal striatum, dHippo or PFC is less defined. Although these regions are key to the development and maintenance of addiction, the neuroadaptation of local CRF2 receptors after heroin exposure in females have not been established previously, as we demonstrate it in this study. One limitation of this study is the fact that these findings are based on females. We excluded males from the study, as we previously reported on the CRF1 antagonist effects on heroin IVSA and related neuroadaptations of CRF1 protein expressions in male rats.

5. Conclusions

In conclusion, we found that antagonism of CRF1 and CRF2 receptors can impair heroin IVSA. Heroin treatment with escalating doses leads to downregulation of CRF1 receptors in the VTA but upregulation in the NAc in female rats. CRF1 expression in the PFC, insula, dorsal striatum, dHippo, HYPTH, amygdala, and SN is not impacted by escalating regimen of heroin exposure. Furthermore, we found that CRF2 receptors become downregulated in the VTA, HYPTH, dHippo but not in the PFC, insula, NAc, striatum, amygdala, or SN following exposure to heroin. As such, the more widespread changes to CRF2 receptors suggest an important, region-specific impact of opioid-related stress and differential resilience to opioid effects.

Highlights.

  • CRF1 and CRF2 receptors are implicated in drug addiction

  • CRF1 and CRF2 receptors play a critical role in heroin self-administration in rats

  • Heroin exposure leads to region-specific neuroadaptations of CRF1 and CRF2 receptors

Acknowledgments:

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1SC3GM130430–03 to RR, the National Institute on Drug Abuse under award number 1R15DA057501 – 01A1 to EG, Colgate University Research Council and in part by National Institute on Drug Abuse Intramural Research Program (to ELG, Neuropsychopharmacology Section, NIDA IRP).

Footnotes

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

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Data Availability:

We are happy to share any protocols or programs that we used to collect these data. The data will be made available upon direct requests to the corresponding author.

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Associated Data

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Data Availability Statement

We are happy to share any protocols or programs that we used to collect these data. The data will be made available upon direct requests to the corresponding author.

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