Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Psychopharmacology (Berl). 2020 Nov 24;238(1):29–40. doi: 10.1007/s00213-020-05652-3

CRF-5-HT interactions in the dorsal raphe nucleus and motivation for stress-induced opioid reinstatement

Chen Li 1,*, Nicholas McCloskey 1,*, Jared Phillips 1, Steven J Simmons 2, Lynn G Kirby 1
PMCID: PMC7796902  NIHMSID: NIHMS1633899  PMID: 33231727

Abstract

Rationale:

The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous data show that stressors can inhibit 5-HT neuronal activity and release by stimulating release of the stress neurohormone corticotropin-releasing factor (CRF) within the serotonergic dorsal raphe nucleus (DRN). The inhibitory effects of CRF on 5-HT DRN neurons are indirect, mediated by CRF-R1 receptors located on GABAergic afferents.

Objectives:

We tested the hypothesis that DRN CRF-R1 receptors contribute to stress-induced reinstatement of morphine conditioned place-preference (CPP). We also examined the role of this circuitry in stress-induced negative affect with 22 kHz distress ultrasonic vocalizations (USVs), which are naturally emitted by rats in response to environmental challenges such as pain, stress, and drug withdrawal.

Methods:

First, we tested if activation of CRF-R1 receptors in the DRN with the CRF-R1-preferring agonist ovine CRF (oCRF) would reinstate morphine CPP and then if blockade of CRF-R1 receptors in the DRN with the CRF-R1 antagonist NBI 35965 would attenuate swim stress-induced reinstatement of morphine CPP. Second, we tested if intra-DRN pretreatment with NBI 35965 would attenuate foot shock stress-induced 22 kHz USVs.

Results:

Intra-DRN injection of oCRF reinstated morphine CPP, while intra-DRN injection of NBI 35965 attenuated swim stress-induced reinstatement. Moreover, intra-DRN pretreatment with NBI 35965 significantly reduced 22 kHz distress calls induced by foot shock.

Conclusions:

These data provide evidence that stress-induced negative affect is mediated by DRN CRF-R1 receptors and may contribute to reinstatement of morphine CPP.

Keywords: Addiction, stressor, relapse, morphine, conditioned place-preference, stress-induced reinstatement, corticotropin-releasing factor (CRF), dorsal raphe, ultrasonic vocalizations, foot shock

1. Introduction

Drug addiction is characterized by repeated relapse to drug use even after a prolonged period of abstinence. Stress is one of the major factors to trigger relapse (Goeders 2003; Sinha 2008). To understand the neurobiological mechanisms underlying stress-induced drug relapse, animal studies have been applied to model relapse in humans. For example, stress, e.g. foot shock or forced swim, can reinstate drug-seeking behavior in animals with a history of conditioned place preference (CPP) and self-administration of drugs (Conrad et al. 2010; Katz and Higgins 2003; Shaham et al. 2003; Staub et al. 2012).

In response to stress, corticotropin-releasing factor (CRF) is released by hypothalamus as the primary neurohormone which activates the hypothalamic-pituitary-adrenal (HPA) axis (Habib et al. 2001; Strohle and Holsboer 2003). The critical role of CRF in stress-induced reinstatement has been identified for several addictive drugs including heroin, cocaine and alcohol (Buffalari et al. 2012; Le et al. 2013; Shaham et al. 1997; Shalev et al. 2010). Interestingly, evidence suggests that the effect of CRF in stress-induced reinstatement may be independent of the HPA axis but instead is related to its actions at extrahypothalamic sites (Erb et al. 1998; Marinelli et al. 2007; Shaham et al. 1997; Shalev et al. 2010), such as the dorsal raphe nucleus (DRN).

The DRN, which contains the majority of the serotonin (5-hydroxytryptamine, 5-HT) neurons projecting to the forebrain (Jacobs and Azmitia 1992), plays an important role in stress-related psychiatric disorders (Baldwin and Rudge 1995; Mann 1999). The role of the DRN in drug-related stress disorders is also supported by studies in our laboratory and others (Ettenberg et al. 2011; Li and Kirby 2016; Staub et al. 2012). For example, data from our laboratory indicate that GABAergic inhibition of the DRN causes reinstatement of morphine CPP whereas disinhibition of the DRN attenuates swim stress-induced reinstatement of morphine CPP (Li et al. 2013).

DRN-5-HT neurons are strongly innervated and regulated by CRF in a bimodal manner, in which activation of different CRF receptor subtypes can have opposing effects on DRN-5-HT neurons (Valentino et al. 2010). Low doses of CRF in the DRN inhibit 5-HT neurons via CRF receptor type1 (CRF-R1), whereas high doses of CRF activate 5-HT neurons via CRF receptor type2 (CRF-R2) (Kirby et al. 2000; Lukkes et al. 2008; Price et al. 1998; Price and Lucki 2001). Although there is evidence that 24 hours after exposure to swim stress DRN-5-HT neurons exhibited increased excitability (Lamy and Beck 2010), acute stress generally inhibits DRN-5-HT activity (Kirby et al. 2000; Price et al. 1998; Price et al. 2002). Anatomical studies showed that DRN GABAergic interneurons receive strong CRF-containing projections (Waselus et al. 2005) and have dense expression of CRF-R1 receptors (Howerton et al. 2014; Lowry et al. 2000; Roche et al. 2003; Valentino et al. 2001). Moreover, electrophysiological studies showed direct evidence that CRF activates CRF-R1 receptors on GABAergic neurons causing increased presynaptic release of GABA onto 5-HT DRN neurons (Kirby et al. 2008). Therefore, we hypothesized that stress-induced activation of CRF-R1 receptors in DRN mediates stress-induced reinstatement. To test our hypothesis, we first examined whether infusion of a CRF-R1-preferring agonist (ovine CRF) (Lovenberg et al. 1995) into DRN would trigger reinstatement of morphine CPP and if infusion of a CRF-R1 antagonist (NBI 35965) could block swim-stress induced reinstatement of morphine CPP.

Stressors potentially motivate drug seeking and reinstatement via their effects on negative affect (Koob 2010; Markou et al. 1998). Given the well-established role of both CRF and 5-HT circuits in affective state, we sought to test affective state in our reinstatement model via measurement of ultrasonic vocalizations (USVs). USVs are naturally emitted calls that rats utilize to communicate aversive or appetitive signals. High frequency (50 kHz) USVs are emitted in response to affiliative or appetitive stimuli such as social interaction and natural or drug reward, whereas low frequency calls (22 kHz) are associated with aversive stimuli such as stress or pain (Burgdorf et al. 2020; Portfors 2007). These latter alarm calls indicate anxiety-like negative affective states in rats (Simola 2015; Simola and Granon 2019). Their utility in studying affective disorders is demonstrated as clinically relevant anxiolytic drugs such as the benzodiazepine diazepam, 5-HT1A receptor agonists buspirone and gepirone, as well as the serotonin-selective reuptake inhibitors citalopram, fluvoxamine, paroxetine, and sertraline all reduce distress vocalization, suggesting a strong link between serotonin activity, negative affect, and 22 kHz USVs (Cullen and Rowan 1994; Jelen et al. 2003). Other behaviors that have been taken to indicate anxiety-like responses in rats, such as freezing and decreased rearing, are associated with USV emission, and drugs that reduce anxious behavior in well-established paradigms like the elevated plus maze or the Vogel choice test likewise attenuate 22 kHz USVs (Bleickardt et al. 2009; Millan et al. 1999; Sánchez 2003a; b; Sánchez et al. 2003). Conversely, administration of the anxiogenic drug pentylenetetrazol increases distress calling (Jelen et al. 2003). Emerging studies suggest that USVs may be useful indicators for different affective states of addiction in rats, e.g. euphoria during drug administration, withdrawal distress and even drug “craving” in reinstatement (Barker et al. 2015; Mahler et al. 2013). Initially, we tried to detect USVs during and after exposure to swim stress or after infusion of intra-DRN oCRF, but were unsuccessful. Previous studies have shown that not all stressors that can reinstate drug-seeking are sufficient to induce 22 kHz USVs (Mahler et al. 2013), indicating that USV vocalization depends on the nature of the stressor. As an alternative strategy, we tested a different stressor, foot shock stress, which can also reinstate drug-seeking (Lu et al. 2003; Mantsch et al. 2016) and reliably induces stress-associated 22 kHz ultrasonic vocalizations (USVs) in rats (Brudzynski 2019; Wöhr et al. 2005). Therefore, to examine whether CRF-5-HT circuits participate in stress-induced negative affect, a potential contributor to relapse vulnerability, we examined whether blocking CRF-R1 receptors in DRN could attenuate foot shock stress-induced 22 kHz USVs in rats.

2. Methods

2.1. Animals

Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 175–200g were housed two per cage at standard temperature (20°C) and humidity (40%) on a 12-h light/dark cycle (day beginning at 7:00am), with ad libitum access to food and water. Following 6–8 days of acclimation, a guide cannula was implanted into the dorsal raphe nucleus (DRN) and animals were then singly housed. Observation and body weight assessment of all subjects ensured proper recovery from surgery. Animal protocols were approved by the Temple University Institutional Animal Care and Use Committee and were conducted in accordance to the National Research Council Guide for the Care and Use of Laboratory Animals.

2.2. Surgery

Rats were anesthetized with an intramuscular ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture and immobilized in a Narishige stereotaxic apparatus to target the DRN at −8.0 mm caudal, −3.5 mm lateral, and −5.0 mm ventral from bregma. The 26-gauge guide cannula (Plastics One, Roanoke, Virginia, USA) was angled at 26° to bypass the sagittal sinus, and secured to the skull with dental cement and two stainless steel screws. An obturator inserted into the cannula maintained patency and prevented infection throughout behavioral testing, which began 5–7 days post-operation.

2.3. Drugs

Morphine (made by the Research Triangle Institute and generously supplied by the National Institute on Drug Abuse) was dissolved in 0.9% saline for subcutaneous administration at 5 mg/kg, a dose selected for its induction of robust, extinguishable, and reinstatable CPP in our previous studies (Li et al. 2013; Staub et al. 2012). To activate CRF-R1 receptors in DRN, the CRF-R1-preferring agonist ovine CRF (oCRF) was prepared in sterile water for infusion into the DRN at a final dosage of 30 ng/0.3 μl. The dose of oCRF was selected because it preferentially activates CRF-R1 receptors in the DRN (Bangasser et al. 2010; Howerton et al. 2014; Lukkes et al. 2008) and it inhibits extracellular 5-HT levels in DRN projection regions (Price and Lucki 2001). To block CRF-R1 receptors, the CRF-R1 antagonist NBI 35965 was prepared in sterile water for DRN infusion at 0.44 ng/0.5 μl. The dose of NBI 35965 was selected because it is 1000-times the Ki for CRF-R1 (Martinez et al. 2004) and prior studies have shown that intra-DRN infusion of 0.44ng NBI 35965 blunted physiological and behavioral stress responses with few confounding side effects (Howerton et al. 2014). All infusions occurred over 1 min, and the infusion needle remained in place for an additional 3 min before removal.

2.4. Conditioned place preference (CPP), extinction and reinstatement

All CPP experimentation was conducted with an unbiased procedure under low-light conditions (maximum 125 lx). The 20×20×40 cm custom-built Plexiglas CPP apparatus consisted of two equally sized chambers, separated with a removable door. The chambers differ in wall design (vertical vs. horizontal black stripes on white background), floor texture (smooth black floor vs. wire elevated 1 cm above a white floor) and cage top/lighting (black top vs. white top backlit with a small spotlight). A preconditioning behavioral test (15 min recorded) ruled out any inherent preference for either of the CPP chambers’ distinct boxes by confirming that the difference in time spent in one side minus that spent in the other was less than 100s, a standard criterion in this paradigm (Herzig and Schmidt 2004; 2005; Li et al. 2013). During the first four days of the conditioning phase animals received a subcutaneous injection of morphine (5 mg/kg) or saline, given alternately in the morning (10am) and afternoon (3pm) sessions, before a 45-min confinement to their drug- or saline-paired side. Injections were counterbalanced for chamber and time of administration. Successful conditioning during the test on day 5 (15 min of free access to both sides) meant that rats spent at least 100 more seconds in their drug-paired side than in the saline-paired side (Herzig and Schmidt 2004; 2005; Li et al. 2013).

The 4-day extinction phase of morphine CPP was structured similarly to conditioning, except that animals received only saline injections in the morning and afternoon sessions on the first two days, and no injections the latter two. In the extinction test (15 minutes of free access to both sides on the 10th day), a difference of less than 100s in time spent in the drug-paired side minus the saline-paired side meant the animal met extinction criteria (Herzig and Schmidt 2004; 2005; Li et al. 2013) and was tested for reinstatement the next day. Rats demonstrating sustained preference for the drug-paired chamber (>100 s difference) received another day of extinction training (both a morning and afternoon 45-min confinement to one side) and were retested for extinction the following day. Once an animal passed an extinction test, it was randomly assigned to a treatment group and its reinstatement test took place the next day, but after three failed retests it was removed from the experiment.

2.4.1. Experiment 1:

Effect of CRF-R1 receptor agonist in DRN on reinstatement of morphine CPP – To test the hypothesis that intra-DRN CRF would trigger reinstatement in subjects with extinguished morphine CPP, oCRF (30 ng/0.3 μl) or vehicle (0.3 μl) was infused through the guide cannula to the DRN 15 min prior to the reinstatement test, which comprised 15 min of free access to both sides of the chamber.

2.4.2. Experiment 2:

Effect of CRF-R1 antagonist in DRN on swim stress-induced reinstatement of morphine CPP – To test the hypothesis that CRF-R1 antagonism would block swim stress-induced reinstatement, NBI 35965 (0.44 ng/0.5 μl) or vehicle (0.5 μl) was infused through the guide cannula to the DRN 15 minutes prior to a forced swim stress (6 min placement into room temperature (21–22 °C) water in a swim tank of 30 cm depth and 20 cm diameter). Following a 20 min drying period, a reinstatement test was administered as described above.

2.5. Ultrasonic Vocalizations (USVs)

To test the hypothesis that stressors elicit a negative affective response through CRF-5-HT circuitry, animals received a foot shock stressor (0.5 mA for 0.5 s) following administration of a CRF-R1 antagonist or vehicle, and their USVs were recorded for analysis. This experiment began with two days of baseline recording, during which animals spent 15 minutes in a sound-attenuated, ventilated chamber (23.5” W × 22” H × 16” D) equipped with a foot shock grid (Med Associates, Inc., St. Albans, VT) and a high-frequency electret condenser microphone UltraMic200K (Dodotronic, Castel Gandolfo, Italy)]. Signals were recorded at 192-kHz and subsequently digitized, amplified, and written to “.wav” files after application of a fast Fourier transform. USV signals were recorded and analyzed with RavenPro software (Cornell Lab of Ornithology, Bioacoustics Research Program, Cornell, NY). During the test on day 3, rats were given an intra-DRN infusion of either NBI 35965 (0.44 ng) or vehicle (0.5 μl) 15 min before placement in the chamber where USVs were recorded for a total of 30 min: 10 min of baseline recording, 5 min of intermittent foot shock stress (5 total shocks at randomized intervals), followed by 15 min of post-stress recording.

2.6. Histology

Following euthanasia by CO2 overdose, 0.7 μl of diluted Evans blue dye was infused through the guide cannula into the DRN over two minutes. Brains were harvested, flash frozen, and sectioned at 100 μm to verify cannula placement (Paxinos and Watson 2005).

2.7. Data Analysis

All data are reported as mean ± SEM and a probability of p < 0.05 was considered significant. All data were initially tested for normality and equal variance prior to statistical analyses. For CPP experiments, the time spent on the drug-paired side minus the unpaired side was analyzed using a two-way mixed-measures ANOVA with phase (conditioning, extinction vs. reinstatement) as a within-subjects factor and reinstatement drug (intraDRN oCRF or NBI 35965 vs. intraDRN vehicle) as a between-subjects factor. Paired-samples t-tests (two-tailed) with Bonferroni correction were conducted as follow-up tests. According to our hypothesis, a pre-planned t-test was conducted to compare reinstatement of morphine CPP between the drug treatment groups since the two groups of animals were treated identically in conditioning and extinction tests. USV data were analyzed using a two-way mixed-measures ANOVA with time as a within-subjects factor and drug treatment (intraDRN NBI 35965 vs intraDRN vehicle) as a between-subjects factor. Paired-samples t-tests (two-tailed) with Bonferroni correction were conducted as follow-up tests. Logarithmic transformation was applied to 50 kHz USV data in order to preserve normal distribution. A constant (+1) was added to these data prior to log transformation in order to avoid null values. Two-way repeated measures ANOVA was applied to these transformed data but Fig. 4d presents the raw data for clarity and consistency with other figures. Statistical analyses were conducted with SigmaPlot 12.5 (Systat Software Inc., San Jose, CA).

3. Results

3.1. Histology

The injection site of the permanent guide cannula, at which each rat received a dose of oCRF, NBI 35965, or saline for behavioral tests, was afterwards confirmed by infusing 0.7 μl of Evans blue dye for histological analysis. The same quantity and procedure were used for each subject to ensure diffusion remained within the DRN’s boundaries. Only the animals with the correct injection placement were included in behavioral analysis (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Schematic representation of injection sites different rostro-caudal levels (−7.3 to −8.3mm from Bregma) of the DRN for CPP and USV experiments. Each dot represents an individual animal. Aq: cerebral aqueduct.

3.2. Conditioned Place Preference (CPP), Extinction and Reinstatement

CPP was used to measure drug reward during conditioning, extinction, and reinstatement tests. Only animals that successfully passed conditioning (82 out of 89, 92%) and extinction (63 out of 79, 80%; 3 additional animals were removed due to cannula failure) tests (see criteria in Materials and Methods section 2.4) were included in the study, as described previously (Staub et al. 2012), because of the focus on the role of DRN CRF-R1 receptors in stress-induced reinstatement. After confirming the placement of injection site, 43 animals with correct intra-DRN injection were included in the behavioral analysis.

3.2.1. Experiment 1:

CRF-R1 receptor agonist in the DRN reinstated morphine CPP – Morphine-conditioned animals spent significantly more time on the drug-paired side than the saline-paired side in the conditioning test, a preference extinguished by repeated extinction training. Intra-DRN injection of oCRF but not vehicle reinstated the preference for the drug-paired side (Fig. 2; vehicle n=11, oCRF n=14). A two-way repeated measures ANOVA revealed main effects of CPP phase [F(2,46)=47.94, p<0.01] and reinstatement drug [F(1,46)=6.00, p<0.05], with a significant interaction [F(2,46)=4.57, p<0.05]. Post hoc t-tests with Bonferroni correction revealed that within the vehicle group, conditioning vs. extinction and conditioning vs. reinstatement were significantly different (p<0.01). Within the oCRF group, conditioning vs. extinction and extinction vs. reinstatement were significantly different (p<0.01). A pre-planned t-test also revealed that within the reinstatement session, the intra-DRN oCRF group was significantly greater than the intra-DRN vehicle group (p<0.01).

Fig. 2.

Fig. 2

Open in a new tab

Morphine CPP, extinction and intra-DRN oCRF-induced reinstatement. Data represent mean ±SEM time spent in drug-paired side minus unpaired side. Intra-DRN injection of oCRF induced reinstatement of previously extinguished morphine CPP (** indicates p<0.01 vs. Intra-DRN vehicle group, ## p<0.01 vs. extinction).

3.2.2. Experiment 2:

CRF-R1 receptor antagonist in the DRN attenuated swim stress-induced reinstatement of morphine CPP – Morphine-conditioned animals spent significantly more time on the drug-paired side than the saline-paired side in the conditioning test, a preference extinguished by repeated extinction training. For animals that received intra-DRN injection of vehicle, swim stress reinstated morphine CPP, an effect not seen in vehicle-treated non-stressed animals in Experiment 1 (Fig. 2). However, swim stress-induced reinstatement was blocked by intra-DRN injection of NBI 35965 (Fig. 3, n=9/group). A two-way repeated measures ANOVA revealed main effects of CPP phase [F(2,32)=9.61, p<0.01] and reinstatement drug [F(1,32)=8.89, p<0.01], with a significant interaction [F(2,32)=9.41, p<0.01]. Post hoc t-tests with Bonferroni correction revealed that within the vehicle group, conditioning vs. extinction and extinction vs. reinstatement were significantly different (p<0.01). Within the NBI 35965 group, conditioning vs. extinction (p<0.05) and conditioning vs. reinstatement (p<0.01) were significantly different. Swim stress-induced reinstatement in the intra-DRN NBI 35965 group was significantly less than in the intra-DRN saline group by pre-planned t-test (p<0.01). There was also a small (n=5) group of NBI 35965-pretreated, swim-stressed animals whose cannula implants missed the DRN and instead delivered infusions into adjacent structures such as the periaqueductal grey (see Supplemental Figure 1). For these anatomical control subjects, mean reinstatement ± SEM was 466.4 ± 42.2 sec which was not statistically significantly different from the vehicle + swim controls (463.3 ± 138.7 sec).

Fig. 3.

Fig. 3

Open in a new tab

Morphine CPP, extinction and swim stress-induced reinstatement. Data represent mean ±SEM time spent in drug-paired side minus unpaired side (n=9/group). Intra-DRN NBI 35965 attenuated swim stress-induced reinstatement of previously extinguished morphine CPP (** indicates p<0.01 vs. Intra-DRN vehicle group, # p<0.05, ## p<0.01 vs. extinction).

3.3. Ultrasonic Vocalizations (USVs)

After confirming the placement of the injection site, 12 animals with correct intra-DRN injection were included in the USV analysis. When exposed to intermittent foot shocks, the intra-DRN vehicle group showed a significant time-dependent elevation of negative affective (22 kHz) USVs. Animals pretreated with intra-DRN NBI 35965 emitted significantly fewer foot shock-induced 22 kHz USVs than their vehicle-infused counterparts (Fig. 4, n=6/group). A two-way repeated measures ANOVA revealed a main effect of pretreatment drug [F(1,50)=8.84, p<0.05] and time [F(5,50)=6.83, p<0.01] for 22 kHz USVs, but only an effect of time [F(5,50)=48.68, p<0.01] for 50 kHz USVs. There were no pretreatment x time interaction effects for either 22 or 50 kHz USVs.

Fig. 4.

Fig. 4

Open in a new tab

Intra-DRN CRF-R1 antagonist NBI 35965 selectively blocks foot shock stress-induced 22 kHz USVs. (a) Example of 22 kHz and 50 kHz USVs, captured from screen shots of spectrographs on short time scale. (b) Representative recordings of 22 kHz USVs for the entire experimental duration in Intra-DRN vehicle vs. NBI 35965 groups. (c) Intra-DRN injection of NBI 35965 attenuated foot shock stress-induced 22 kHz USVs compared with vehicle group. Data represent mean ±SEM number of USVs emitted during a 5-min time bin. (* indicates a main effect of drug treatment, p<0.05). (d) intra-DRN injection of NBI 35965 had no effects on 50 kHz USVs primarily emitted during the baseline of the experiment.

4. Discussion

This study demonstrates that CRF transmission in the DRN-5-HT system is critical for stress-induced reinstatement of morphine CPP. Intra-DRN injection of a CRF-R1 receptor agonist induced reinstatement of morphine CPP, while intra-DRN injection of a CRF-R1 receptor antagonist blocked swim stress-induced reinstatement. Additionally, DRN CRF-R1 receptors mediate stress-induced negative affect, as measured by foot shock stress-induced 22 kHz distress calls. Together with prior evidence that DRN CRF-R1 receptors largely inhibit 5-HT output (Kirby et al. 2008; Kirby et al. 2000; Lukkes et al. 2008), these data suggest that stress engages CRF afferents to the DRN, inhibiting 5-HT neurons which leads to a negative affective state that can motivate opioid reinstatement.

Exogenous administration of CRF induces reinstatement for many major classes of drugs of abuse and is thought to be implicated in allostatic changes leading to relapse vulnerability (Buffalari et al. 2012; Koob 1999; Shaham et al. 1997). Conversely, CRF antagonists block stress-induced reinstatement in animals with a history of exposure to drugs of abuse (Heilig and Koob 2007; Shaham et al. 1997). CRF has an important role in the aversive states of withdrawal for opiates, psychostimulants, alcohol, and nicotine (Shalev et al. 2010), which is consistent with our hypothesis that this neurohormone elicits negative affect that motivates relapse.

Interestingly, the effect of CRF on 5-HT cells is not always inhibitory (Kirby et al. 2000; Lowry et al. 2000) and to understand the complexity of the system it is necessary to understand the topographical nature of the DRN which has been the topic of several detailed reviews (Abrams et al. 2004; Okaty et al. 2019) including the perspective of this topography in the context of stress and CRF (Fox and Lowry 2013; Valentino and Commons 2005). GABA neurons are expressed throughout the rostro-caudal extent of the DRN and either flank 5-HT neurons in midline DRN or intermingle with 5-HT neurons in the lateral wings (Day et al. 2004). Ultrastructural studies of the DRN have shown that CRF-R1 receptors are expressed in greater numbers on DRN GABA terminals than 5-HT neurons (Waselus et al. 2005), including in the ventromedial subdivision targeted in this study. Electrophysiology studies in the ventromedial DRN have also demonstrated CRF-R1-mediated elevation of GABA release onto 5-HT neurons in vitro (Kirby et al. 2008) and CRF-R1-mediated inhibition of 5-HT DRN neuronal activity in vivo (Kirby et al. 2000). By contrast, CRF-R2 localizes primarily to 5-HT DRN neurons, particularly in mid-rostral DRN (Day et al. 2004), and largely stimulates 5-HT output (Kirby et al. 2008; Lukkes et al. 2008). These anatomical and physiological studies in combination with the receptor selectivity and doses of the ligands used in the current study support CRF-R1-mediated suppression of 5-HT output as the basis of our behavioral and USV findings. However, CRF modulation of 5-HT DRN neurons also depends on the nature of the stress exposure (Snyder et al. 2015). While DRN GABA neurons expressing CRF-R1 are engaged by stressors such as forced swim stress (Roche et al. 2003), DRN CRF-R2, particularly at more caudal DRN levels, has been associated with uncontrollable stressors such as learned helplessness (Hammack et al. 2002; Hammack et al. 2003). After repeated exposure to stress, the prevalence of receptor subtypes in the plasma membrane of 5-HT DRN neurons shifts from predominantly CRF-R1 to CRF-R2 (Waselus et al. 2009; Wood et al. 2013). Together, these data indicate that CRF can dynamically regulate 5-HT output and potentially mood state, allowing for shifts in active vs passive coping responses to acute vs chronic stressors, respectively (Hupalo et al. 2019; Valentino et al. 2010; Waselus et al. 2011). Ultimately, the effects of CRF on the 5-HT DRN system and the role of these interactions in stress-related behaviors varies depending on the dose of CRF tested, the CRF receptor subtype that is engaged, the neurochemical nature of the DRN neuron expressing that receptor and the subpopulation of 5-HT neurons that is impacted.

Our current findings are consistent with previous studies demonstrating the role of 5-HT in reinstatement in animals with drug history (Filip et al. 2010; Kirby et al. 2011; Müller and Homberg 2015). For example, stimulation of the 5-HT system with 5-HT-selective reuptake inhibitors or 5-HT releasing agents suppresses both cue-elicited cocaine reinstatement (Baker et al. 2001; Burmeister et al. 2003) and also stress-induced reinstatement of alcohol seeking behavior in rats (Le et al. 2006; Le et al. 1999). Although only a few studies have investigated the role of the sources of the serotonergic projections in drug reinstatement, our laboratory and others have implicated both dorsal and median raphe in stress-induced relapse (Land et al. 2009; Le et al. 2013; Le et al. 2008; Le et al. 2002; Li et al. 2013). Further examination of DRN circuits and drug reinstatement indicate that DRN GABA neurons have an important role in this process. Whereas GABAA agonists reinstate previously extinguished morphine CPP, antagonists block stress-induced reinstatement (Li et al. 2013). Additionally, our prior studies have identified a general neuroadaptation of 5-HT neurons that is linked to stress-induced reinstatement of both opiates and psychostimulants (Li and Kirby 2016; Staub et al. 2012). We observed sensitization of 5-HT neurons to GABAergic inhibition that was specific to animals demonstrating stress-induced reinstatement to either previously extinguished morphine CPP or cocaine self-administration. Consequently, we hypothesize that sensitization of 5-HT neurons to GABAergic inhibition contributes to vulnerability to stress-induced reinstatement. Our model (Fig. 5) posits that stress engages CRF afferents to the DRN, stimulating CRF-R1 on GABA neurons, inhibiting 5-HT output. Hypofunction of the 5-HT system and the resulting negative affect motivates reinstatement.

Fig. 5.

Fig. 5

Open in a new tab

Model of the interaction between CRF and 5-HT circuits and its involvement in negative affect and stress-induced relapse.

Rats naturally emit ultrasonic vocalizations in response to environmental stimuli, generally with 22 kHz calls indicating distress or negative affect and 50 kHz calls representing reward or positive affect (Barker et al. 2015; Brudzynski 2019; Burgdorf et al. 2020; Portfors 2007). In the current study, foot shock stress elicited 22 kHz distress calls, but intra-DRN pretreatment with the CRF-R1 selective antagonist NBI 35965 significantly attenuated that response, suggesting a critical role of DRN CRF-R1 in stress-induced negative affect. This finding is consistent with prior studies demonstrating that rat pups’ emission of USV distress calls induced by maternal separation stress were attenuated by systemic administration of a CRF-R1 receptor antagonist as well as by a selective serotonin reuptake inhibitor (Iijima and Chaki 2005; Ise et al. 2008; Kehne et al. 2000) . Distress signals can also be elicited by social defeat stress, a model in which an animal is systematically attacked and defeated by a dominant conspecific male. Marini et al. (2006) demonstrated that social defeat both increases 22 kHz USVs and increases mRNA for CRF, CRF-R1, and CRF-binding protein in the hippocampus. Moreover, CRF-R1 antagonist can attenuate the retrieval process of fear-conditioned ultrasonic vocalization in rats (Kikusui et al. 2000). Consequently, CRF signaling through CRF-R1 has been linked to negative affect in prior studies and our current work identifies CRF-R1 in the DRN as a key site mediating some of those effects.

Some limitations of the present study must be mentioned, and further research is required to fully characterize the role of CRF-5-HT circuits in stress-induced reinstatement. First, the effect of NBI 35965 was not tested under reinstatement conditions in the absence of a stressor, allowing for the possibility that blockade of DRN CRF-R1 receptors that are occupied by endogenous CRF under normal physiological conditions were responsible for NBI 35965’s effects on reinstatement. However, previous drug reinstatement studies that tested CRF-R1-selective antagonists under non-stress conditions have shown only marginal impacts of the antagonist itself on reinstatement of previously extinguished cocaine CPP (Kreibich and Blendy 2004) or alcohol self-administration (Funk et al. 2014). Therefore, it is likely that NBI 35965’s effects in this study were largely mediated by its blockade of swim stress-induced release of endogenous CRF within the DRN that bind to CRF-R1 (Price et al. 2002). Second, intra-DRN infusion of the CRF-R1 receptor agonist and antagonist would impact not only 5-HT neurons but any other cell types and neuronal fibers in the DRN expressing those receptors. A conditional knockout approach in future studies will allow suppression or activation of CRF-R1 receptors in only DRN-5-HT neurons to help resolve this limitation. Third, the DRN is known to have a complex topography in its multiple anatomic and functional subdivisions (i.e. ventromedial, dorsomedial, lateral wings) with distinctive afferent and efferent connections, neurochemical and neurophysiological properties (Abrams et al. 2004; Calizo et al. 2011; Waselus et al. 2011). Diffusion of the infused CRF-R1-selective ligands prevented selective targeting of these subdivisions. Smaller infusion volumes directed to more precise surgical sites in future studies would help to determine each subregion’s specific contribution to stress-induced reinstatement. Fourth, the bimodal influence of CRF on 5-HT DRN neurons through differentially expressed receptor subtypes needs to be investigated. The present study focused on CRF-R1 receptors, known to be expressed on GABA terminals (Waselus et al. 2005). CRF-R1 effects would influence 5-HT output indirectly, mediated by GABAA receptors expressed on 5-HT neurons that have previously shown to be both necessary and sufficient for stress-induced reinstatement of morphine CPP (Li et al. 2013). Future work will need to compare these effects to those of CRF-R2 receptor-selective agonists and antagonists. Fifth, while CPP model reveals the hedonic value of addictive drugs, self-administration better represents voluntary drug-seeking behavior in humans (Sanchis-Segura and Spanagel 2006). Initial studies using stress-induced heroin self-administration have yielded preliminary data to suggest that 5-HT neurons show sensitization to GABAergic inhibition in response to stress-induced heroin reinstatement (unpublished observations) that is consistent with prior publications from stress-induced reinstatement of both cocaine self-administration (Li and Kirby 2016) and morphine CPP (Staub et al. 2012). Sixth, this study focused on USVs in the 50 kHz range that reflect positive affect and USVs in the 22 kHz range that reflect negative affect. While these two USV categories have been the most extensively characterized in the literature (Burgdorf et al. 2020; Portfors 2007), Mahler et al. (2013) have identified multiple subtypes of calls in a self-administration paradigm across a range of frequencies, whose patterns of expression throughout a stress-induced reinstatement paradigm suggest an expanded vocal repertoire that is capable of signaling more specific affective states and should be explored in future studies. Last, while this and other studies support the connection between 22kHz USVs, serotonin and negative affect (Cullen and Rowan 1994; Jelen et al. 2003), the inability of forced swim stress to elicit vocalizations necessitated the use of different stressors in the CPP and USV paradigms, so fully reconciling that technical incongruity in our behavioral and affective measures requires additional investigation employing the same stressor to provoke both distress calls and reinstatement. Ongoing work with foot shock stress-induced reinstatement of heroin self-administration will allow us to examine USVs and behavioral responses to the same stressor and will further test the causal role of CRF-GABA-5-HT interactions of the DRN in this model.

The current study along with previous work supports the hypothesis that stressors promote negative affect and trigger opioid reinstatement by engaging CRF afferents to 5-HT DRN neurons. While serotonergic and CRF ligands have shown limited clinical efficacy in the treatment of substance abuse or relapse prevention to date (Greenwald 2018; Indave et al. 2016), the current preclinical data suggest additional possibilities for targeting CRF-5-HT systems alone or in combination in the search for novel targets for the treatment of opioid addiction and the prevention of opioid relapse.

Supplementary Material

supp fig

Supplemental Fig. 1 Schematic representation of injection sites outside of the DRN for CPP experiments. Each dot represents an individual animal. Aq: cerebral aqueduct.

Acknowledgement:

supported by NIH grants R01 DA045771, R01 DA020126 and P30 DA013429

Footnotes

Conflict of interest:

All authors declare no conflict of interests and have no disclosures

References

  1. Abrams JK, Johnson PL, Hollis JH, Lowry CA (2004) Anatomic and functional topography of the dorsal raphe nucleus. Annals of the New York Academy of Sciences 1018: 46–57. [DOI] [PubMed] [Google Scholar]
  2. Baker DA, Tran-Nguyen TL, Fuchs RA, Neisewander JL (2001) Influence of individual differences and chronic fluoxetine treatment on cocaine-seeking behavior in rats. Psychopharmacology (Berl) 155: 18–26. [DOI] [PubMed] [Google Scholar]
  3. Baldwin D, Rudge S (1995) The role of serotonin in depression and anxiety. Int Clin Psychopharmacol 9 Suppl 4: 41–5. [DOI] [PubMed] [Google Scholar]
  4. Bangasser DA, Curtis A, Reyes BA, Bethea TT, Parastatidis I, Ischiropoulos H, Van Bockstaele EJ, Valentino RJ (2010) Sex differences in corticotropin-releasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology. Molecular Psychiatry 15: 877, 896–877, 904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barker DJ, Simmons SJ, West MO (2015) Ultrasonic Vocalizations as a Measure of Affect in Preclinical Models of Drug Abuse: A Review of Current Findings. CurrNeuropharmacol 13: 193–210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bleickardt CJ, Mullins DE, Macsweeney CP, Werner BJ, Pond AJ, Guzzi MF, Martin FD, Varty GB, Hodgson RA (2009) Characterization of the V1a antagonist, JNJ-17308616, in rodent models of anxiety-like behavior. Psychopharmacology (Berl) 202: 711–8. [DOI] [PubMed] [Google Scholar]
  7. Brudzynski SM (2019) Emission of 22 kHz vocalizations in rats as an evolutionary equivalent of human crying: Relationship to depression. Behav Brain Res 363: 1–12. [DOI] [PubMed] [Google Scholar]
  8. Buffalari DM, Baldwin CK, Feltenstein MW, See RE (2012) Corticotrophin releasing factor (CRF) induced reinstatement of cocaine seeking in male and female rats. Physiol Behav 105: 209–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burgdorf JS, Brudzynski SM, Moskal JR (2020) Using rat ultrasonic vocalization to study the neurobiology of emotion: from basic science to the development of novel therapeutics for affective disorders. Curr Opin Neurobiol 60: 192–200. [DOI] [PubMed] [Google Scholar]
  10. Burmeister JJ, Lungren EM, Neisewander JL (2003) Effects of fluoxetine and d-fenfluramine on cocaine-seeking behavior in rats. Psychopharmacology (Berl) 168: 146–154. [DOI] [PubMed] [Google Scholar]
  11. Calizo LH, Akanwa A, Ma X, Pan YZ, Lemos JC, Craige C, Heemstra LA, Beck SG (2011) Raphe serotonin neurons are not homogenous: electrophysiological, morphological and neurochemical evidence. Neuropharmacology 61: 524–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conrad KL, McCutcheon JE, Cotterly LM, Ford KA, Beales M, Marinelli M (2010) Persistent increases in cocaine-seeking behavior after acute exposure to cold swim stress. Biological Psychiatry 68: 303–305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cullen WK, Rowan MJ (1994) Gepirone and 1-(2-pyrimidinyl)-piperazine-induced reduction of aversively evoked ultrasonic vocalisation in the rat. Pharmacol Biochem Behav 48: 301–6. [DOI] [PubMed] [Google Scholar]
  14. Day HE, Greenwood BN, Hammack SE, Watkins LR, Fleshner M, Maier SF, Campeau S (2004) Differential expression of 5HT-1A, alpha 1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic, gamma-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus. JComp Neurol 474: 364–378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Erb S, Shaham Y, Stewart J (1998) The role of corticotropin-releasing factor and corticosterone in stress- and cocaine-induced relapse to cocaine seeking in rats. JNeurosci 18: 5529–5536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ettenberg A, Ofer OA, Mueller CL, Waldroup S, Cohen A, Ben-Shahar O (2011) Inactivation of the dorsal raphe nucleus reduces the anxiogenic response of rats running an alley for intravenous cocaine. Pharmacol Biochem Behav 97: 632–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Filip M, Alenina N, Bader M, Przegalinski E (2010) Behavioral evidence for the significance of serotoninergic (5-HT) receptors in cocaine addiction. AddictBiol 15: 227–249. [DOI] [PubMed] [Google Scholar]
  18. Fox JH, Lowry CA (2013) Corticotropin-releasing factor-related peptides, serotonergic systems, and emotional behavior. Front Neurosci 7: 169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Funk D, Coen K, Lê AD (2014) The role of kappa opioid receptors in stress-induced reinstatement of alcohol seeking in rats. Brain Behav 4: 356–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goeders NE (2003) The impact of stress on addiction. Eur Neuropsychopharmacol 13: 435–41. [DOI] [PubMed] [Google Scholar]
  21. Greenwald MK (2018) Anti-stress neuropharmacological mechanisms and targets for addiction treatment: A translational framework. Neurobiol Stress 9: 84–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Habib KE, Gold PW, Chrousos GP (2001) Neuroendocrinology of stress. Endocrinol Metab Clin North Am 30: 695–728; vii-viii. [DOI] [PubMed] [Google Scholar]
  23. Hammack SE, Richey KJ, Schmid MJ, LoPresti ML, Watkins LR, Maier SF (2002) The role of corticotropin-releasing hormone in the dorsal raphe nucleus in mediating the behavioral consequences of uncontrollable stress. JNeurosci 22: 1020–1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hammack SE, Schmid MJ, LoPresti ML, Der-Avakian A, Pellymounter MA, Foster AC, Watkins LR, Maier SF (2003) Corticotropin releasing hormone type 2 receptors in the dorsal raphe nucleus mediate the behavioral consequences of uncontrollable stress. JNeurosci 23: 1019–1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heilig M, Koob GF (2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci 30: 399–406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Herzig V, Schmidt WJ (2004) Effects of MPEP on locomotion, sensitization and conditioned reward induced by cocaine or morphine. Neuropharmacology 47: 973–984. [DOI] [PubMed] [Google Scholar]
  27. Herzig V, Schmidt WJ (2005) Repeated-testing of place preference expression for evaluation of anti-craving-drug effects. AminoAcids 28: 309–317. [DOI] [PubMed] [Google Scholar]
  28. Howerton AR, Roland AV, Fluharty JM, Marshall A, Chen A, Daniels D, Beck SG, Bale TL (2014) Sex differences in corticotropin-releasing factor receptor-1 action within the dorsal raphe nucleus in stress responsivity. Biological Psychiatry 75: 873–883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hupalo S, Bryce CA, Bangasser DA, Berridge CW, Valentino RJ, Floresco SB (2019) Corticotropin-Releasing Factor (CRF) circuit modulation of cognition and motivation. Neurosci Biobehav Rev 103: 50–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Iijima M, Chaki S (2005) Separation-induced ultrasonic vocalization in rat pups: further pharmacological characterization. Pharmacol Biochem Behav 82: 652–7. [DOI] [PubMed] [Google Scholar]
  31. Indave BI, Minozzi S, Pani PP, Amato L (2016) Antipsychotic medications for cocaine dependence. Cochrane Database Syst Rev 3: CD006306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ise S, Nagano N, Okuda S, Ohta H (2008) Corticotropin-releasing factor modulates maternal separation-induced ultrasonic vocalization in rat pups via activation of CRF1 receptor. Brain Res 1234: 59–65. [DOI] [PubMed] [Google Scholar]
  33. Jacobs BL, Azmitia EC (1992) Structure and function of the brain serotonin system. Physiological Reviews 72: 165–229. [DOI] [PubMed] [Google Scholar]
  34. Jelen P, Soltysik S, Zagrodzka J (2003) 22-kHz ultrasonic vocalization in rats as an index of anxiety but not fear: behavioral and pharmacological modulation of affective state. Behav Brain Res 141: 63–72. [DOI] [PubMed] [Google Scholar]
  35. Jolas T, Aghajanian GK (1997) Opioids suppress spontaneous and NMDA-induced inhibitory postsynaptic currents in the dorsal raphe nucleus of the rat in vitro. Brain Research 755: 229–245. [DOI] [PubMed] [Google Scholar]
  36. Katz JL, Higgins ST (2003) The validity of the reinstatement model of craving and relapse to drug use. Psychopharmacology (Berl) 168: 21–30. [DOI] [PubMed] [Google Scholar]
  37. Kehne JH, Coverdale S, McCloskey TC, Hoffman DC, Cassella JV (2000) Effects of the CRF(1) receptor antagonist, CP 154,526, in the separation-induced vocalization anxiolytic test in rat pups. Neuropharmacology 39: 1357–67. [DOI] [PubMed] [Google Scholar]
  38. Kikusui T, Takeuchi Y, Mori Y (2000) Involvement of corticotropin-releasing factor in the retrieval process of fear-conditioned ultrasonic vocalization in rats. Physiol Behav 71: 323–8. [DOI] [PubMed] [Google Scholar]
  39. Kirby LG, Freeman-Daniels E, Lemos JC, Nunan JD, Lamy C, Akanwa A, Beck SG (2008) Corticotropin-releasing factor increases GABA synaptic activity and induces inward current in 5-hydroxytryptamine dorsal raphe neurons. J Neurosci 28: 12927–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kirby LG, Rice KC, Valentino RJ (2000) Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus. Neuropsychopharmacology 22: 148–62. [DOI] [PubMed] [Google Scholar]
  41. Kirby LG, Zeeb FD, Winstanley CA (2011) Contributions of serotonin in addiction vulnerability. Neuropharmacology 61: 421–432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Koob GF (1999) Stress, corticotropin-releasing factor, and drug addiction. Annals of the New York Academy of Sciences 897: 27–45. [DOI] [PubMed] [Google Scholar]
  43. Koob GF (2010) The role of CRF and CRF-related peptides in the dark side of addiction. Brain Research 1314: 3–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kreibich AS, Blendy JA (2004) cAMP response element-binding protein is required for stress but not cocaine-induced reinstatement. JNeurosci 24: 6686–6692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Lamy CM, Beck SG (2010) Swim stress differentially blocks CRF receptor mediated responses in dorsal raphe nucleus. Psychoneuroendocrinology 35: 1321–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Land BB, Bruchas MR, Schattauer S, Giardino WJ, Aita M, Messinger D, Hnasko TS, Palmiter RD, Chavkin C (2009) Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. ProcNatlAcadSciUSA 106: 19168–19173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Le AD, Funk D, Coen K, Li Z, Shaham Y (2013) Role of corticotropin-releasing factor in the median raphe nucleus in yohimbine-induced reinstatement of alcohol seeking in rats. AddictBiol 18: 448–451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Le AD, Funk D, Harding S, Juzytsch W, Fletcher PJ, Shaham Y (2006) Effects of dexfenfluramine and 5-HT3 receptor antagonists on stress-induced reinstatement of alcohol seeking in rats. Psychopharmacology (Berl) 186: 82–92. [DOI] [PubMed] [Google Scholar]
  49. Le AD, Funk D, Harding S, Juzytsch W, Li Z, Fletcher PJ (2008) Intra-median raphe nucleus (MRN) infusions of muscimol, a GABA-A receptor agonist, reinstate alcohol seeking in rats: role of impulsivity and reward. Psychopharmacology (Berl) 195: 605–615. [DOI] [PubMed] [Google Scholar]
  50. Le AD, Harding S, Juzytsch W, Fletcher PJ, Shaham Y (2002) The role of corticotropin-releasing factor in the median raphe nucleus in relapse to alcohol. Journal of Neuroscience 22: 7844–7849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Le AD, Poulos CX, Harding S, Watchus J, Juzytsch W, Shaham Y (1999) Effects of naltrexone and fluoxetine on alcohol self-administration and reinstatement of alcohol seeking induced by priming injections of alcohol and exposure to stress. Neuropsychopharmacology 21: 435–44. [DOI] [PubMed] [Google Scholar]
  52. Li C, Kirby LG (2016) Effects of cocaine history on postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats. European Neuropsychopharmacology 26: 45–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Li C, Staub DR, Kirby LG (2013) Role of GABA receptors in dorsal raphe nucleus in stress-induced reinstatement of morphine-conditioned place preference in rats. Psychopharmacology (Berl) 230: 537–545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdorf T (1995) Cloning and characterization of a functionally distinct corticotropin- releasing factor receptor subtype from rat brain. Proceedings of the National Academy of Sciences of the United States of America 92: 836–840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Lowry CA, Rodda JE, Lightman SL, Ingram CD (2000) Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system. JNeurosci 20: 7728–7736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Lu L, Shepard JD, Hall FS, Shaham Y (2003) Effect of environmental stressors on opiate and psychostimulant reinforcement, reinstatement and discrimination in rats: a review. Neuroscience & Biobehavioral Reviews 27: 457–491. [DOI] [PubMed] [Google Scholar]
  57. Lukkes JL, Forster GL, Renner KJ, Summers CH (2008) Corticotropin-releasing factor 1 and 2 receptors in the dorsal raphe differentially affect serotonin release in the nucleus accumbens. European Journal of Pharmacology 578: 185–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Mahler SV, Moorman DE, Feltenstein MW, Cox BM, Ogburn KB, Bachar M, McGonigal JT, Ghee SM, See RE (2013) A rodent “self-report” measure of methamphetamine craving? Rat ultrasonic vocalizations during methamphetamine self-administration, extinction, and reinstatement. Behav Brain Res 236: 78–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Mann JJ (1999) Role of the serotonergic system in the pathogenesis of major depression and suicidal behavior. Neuropsychopharmacology 21: 99S–105S. [DOI] [PubMed] [Google Scholar]
  60. Mantsch JR, Baker DA, Funk D, Le AD, Shaham Y (2016) Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress. Neuropsychopharmacology 41: 335–356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Marinelli PW, Funk D, Juzytsch W, Harding S, Rice KC, Shaham Y, Le AD (2007) The CRF1 receptor antagonist antalarmin attenuates yohimbine-induced increases in operant alcohol self-administration and reinstatement of alcohol seeking in rats. Psychopharmacology (Berl) 195: 345–55. [DOI] [PubMed] [Google Scholar]
  62. Marini F, Pozzato C, Andreetta V, Jansson B, Arban R, Domenici E, Carboni L (2006) Single exposure to social defeat increases corticotropin-releasing factor and glucocorticoid receptor mRNA expression in rat hippocampus. Brain Res 1067: 25–35. [DOI] [PubMed] [Google Scholar]
  63. Markou A, Kosten TR, Koob GF (1998) Neurobiological similarities in depression and drug dependence: a self-medication hypothesis. Neuropsychopharmacology 18: 135–174. [DOI] [PubMed] [Google Scholar]
  64. Martinez V, Wang L, Rivier J, Grigoriadis D, Tache Y (2004) Central CRF, urocortins and stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors delays gastric transit in mice. JPhysiol 556: 221–234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Millan MJ, Brocco M, Gobert A, Schreiber R, Dekeyne A (1999) S-16924 [(R)-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]- pyrrolidin-3yl]-1-(4-fluorophenyl)-ethanone], a novel, potential antipsychotic with marked serotonin1A agonist properties: III. Anxiolytic actions in comparison with clozapine and haloperidol. J Pharmacol Exp Ther 288: 1002–14. [PubMed] [Google Scholar]
  66. Müller CP, Homberg JR (2015) The role of serotonin in drug use and addiction. Behav Brain Res 277: 146–92. [DOI] [PubMed] [Google Scholar]
  67. Okaty BW, Commons KG, Dymecki SM (2019) Embracing diversity in the 5-HT neuronal system. Nat Rev Neurosci 20: 397–424. [DOI] [PubMed] [Google Scholar]
  68. Paxinos G, Watson C (2005) The Rat Brain in Stereotaxic Coordinates. Academic Press, New York [Google Scholar]
  69. Portfors CV (2007) Types and functions of ultrasonic vocalizations in laboratory rats and mice. J Am Assoc Lab Anim Sci 46: 28–34. [PubMed] [Google Scholar]
  70. Price ML, Curtis AL, Kirby LG, Valentino RJ, Lucki I (1998) Effects of corticotropin-releasing factor on brain serotonergic activity. Neuropsychopharmacology 18: 492–502. [DOI] [PubMed] [Google Scholar]
  71. Price ML, Kirby LG, Valentino RJ, Lucki I (2002) Evidence for corticotropin-releasing factor regulation of serotonin in the lateral septum during acute swim stress: adaptation produced by repeated swimming. Psychopharmacology (Berl) 162: 406–414. [DOI] [PubMed] [Google Scholar]
  72. Price ML, Lucki I (2001) Regulation of serotonin release in the lateral septum and striatum by corticotropin-releasing factor. JNeurosci 21: 2833–2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Roche M, Commons KG, Peoples A, Valentino RJ (2003) Circuitry underlying regulation of the serotonergic system by swim stress. JNeurosci 23: 970–977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Sanchis-Segura C, Spanagel R (2006) Behavioural assessment of drug reinforcement and addictive features in rodents: an overview. Addict Biol 11: 2–38. [DOI] [PubMed] [Google Scholar]
  75. Shaham Y, Funk D, Erb S, Brown TJ, Walker CD, Stewart J (1997) Corticotropin-releasing factor, but not corticosterone, is involved in stress-induced relapse to heroin-seeking in rats. JNeurosci 17: 2605–2614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Shaham Y, Shalev U, Lu L, De Wit H, Stewart J (2003) The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology (Berl) 168: 3–20. [DOI] [PubMed] [Google Scholar]
  77. Shalev U, Erb S, Shaham Y (2010) Role of CRF and other neuropeptides in stress-induced reinstatement of drug seeking. Brain Res 1314: 15–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Simola N (2015) Rat Ultrasonic Vocalizations and Behavioral Neuropharmacology: From the Screening of Drugs to the Study of Disease. CurrNeuropharmacol 13: 164–179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Simola N, Granon S (2019) Ultrasonic vocalizations as a tool in studying emotional states in rodent models of social behavior and brain disease. Neuropharmacology 159: 107420. [DOI] [PubMed] [Google Scholar]
  80. Sinha R (2008) Chronic stress, drug use, and vulnerability to addiction. Ann N Y Acad Sci 1141: 105–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Snyder KP, Hill-Smith TE, Lucki I, Valentino RJ (2015) Corticotropin-releasing Factor in the Rat Dorsal Raphe Nucleus Promotes Different Forms of Behavioral Flexibility Depending on Social Stress History. Neuropsychopharmacology 40: 2517–2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Staub DR, Lunden JW, Cathel AM, Dolben EL, Kirby LG (2012) Morphine history sensitizes postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats. Psychoneuroendocrinology 37: 859–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Staub DR, Lunden JW, Freeman-Daniels EL, Kirby LG (2008) Stress-induced opiate reinstatement: Electrophysiological studies in dorsal raphe serotonin neurons. Society for Neuroscience Abstracts: 767.22. [Google Scholar]
  84. Strohle A, Holsboer F (2003) Stress responsive neurohormones in depression and anxiety. Pharmacopsychiatry 36 Suppl 3: S207–14. [DOI] [PubMed] [Google Scholar]
  85. Sánchez C (2003a) R-citalopram attenuates anxiolytic effects of escitalopram in a rat ultrasonic vocalisation model. Eur J Pharmacol 464: 155–8. [DOI] [PubMed] [Google Scholar]
  86. Sánchez C (2003b) Stress-induced vocalisation in adult animals. A valid model of anxiety? Eur J Pharmacol 463: 133–43. [DOI] [PubMed] [Google Scholar]
  87. Sánchez C, Bergqvist PB, Brennum LT, Gupta S, Hogg S, Larsen A, Wiborg O (2003) Escitalopram, the S-(+)-enantiomer of citalopram, is a selective serotonin reuptake inhibitor with potent effects in animal models predictive of antidepressant and anxiolytic activities. Psychopharmacology (Berl) 167: 353–62. [DOI] [PubMed] [Google Scholar]
  88. Tao R, Auerbach SB (2005) mu-Opioids disinhibit and kappa-opioids inhibit serotonin efflux in the dorsal raphe nucleus. Brain Res 1049: 70–9. [DOI] [PubMed] [Google Scholar]
  89. Valentino RJ, Commons KG (2005) Peptides that fine-tune the serotonin system. Neuropeptides 39: 1–8. [DOI] [PubMed] [Google Scholar]
  90. Valentino RJ, Liouterman L, Van Bockstaele EJ (2001) Evidence for regional heterogeneity in corticotropin-releasing factor interactions in the dorsal raphe nucleus. JComp Neurol 435: 450–463. [DOI] [PubMed] [Google Scholar]
  91. Valentino RJ, Lucki I, Van Bockstaele E (2010) Corticotropin-releasing factor in the dorsal raphe nucleus: Linking stress coping and addiction. Brain Res 1314: 29–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Waselus M, Nazzaro C, Valentino RJ, Van Bockstaele EJ (2009) Stress-induced redistribution of corticotropin-releasing factor receptor subtypes in the dorsal raphe nucleus. Biological Psychiatry 66: 76–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Waselus M, Valentino RJ, Van Bockstaele EJ (2005) Ultrastructural evidence for a role of gamma-aminobutyric acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system. JComp Neurol 482: 155–165. [DOI] [PubMed] [Google Scholar]
  94. Waselus M, Valentino RJ, Van Bockstaele EJ (2011) Collateralized dorsal raphe nucleus projections: a mechanism for the integration of diverse functions during stress. Journal of Chemical Neuroanatomy 41: 266–280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Wood SK, Zhang XY, Reyes BA, Lee CS, Van Bockstaele EJ, Valentino RJ (2013) Cellular adaptations of dorsal raphe serotonin neurons associated with the development of active coping in response to social stress. Biological Psychiatry 73: 1087–1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Wöhr M, Borta A, Schwarting RK (2005) Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: a dose-response study in the rat. Neurobiol Learn Mem 84: 228–40. [DOI] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

supp fig

Supplemental Fig. 1 Schematic representation of injection sites outside of the DRN for CPP experiments. Each dot represents an individual animal. Aq: cerebral aqueduct.

NIHMS1633899-supplement-supp_fig.tif (782.8KB, tif)

RESOURCES