Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 15.
Published in final edited form as: Neuropharmacology. 2024 Jan 3;246:109832. doi: 10.1016/j.neuropharm.2023.109832

Requisite role of dorsal raphé in contextual cocaine-memory reconsolidation

JL Ritchie a, S Qi a, RJ Christian a, MJ Greenwood a, HI Grenz a, SE Swatzell a, PJ Krych a, RA Fuchs a,b,*
PMCID: PMC10901441  NIHMSID: NIHMS1967081  PMID: 38176535

Abstract

Memory reconsolidation is a process by which labile drug memories are restabilized in long-term memory stores, permitting their enduring control over drug-seeking behaviors. In the present study, we investigated the involvement of the dorsal raphé nuclei (DRN) in cocaine-memory reconsolidation. Sprague-Dawley rats (male, female) were trained to self-administer cocaine in a distinct environmental context to establish contextual drug memories. They then received extinction training in a different context. Next, the rats were re-exposed to the cocaine-predictive context for 15 minutes to reactivate their cocaine memories or remained in their home cages (no-reactivation control). Memory reactivation was sufficient to increase c-Fos expression, an index of neuronal activation, in the DRN, but not in the medial raphé nuclei, during reconsolidation, compared to no reactivation. To determine whether DRN neuronal activity was necessary for cocaine-memory reconsolidation, rats received intra-DRN baclofen plus muscimol (BM; GABAB/A agonists) or vehicle microinfusions immediately after or 6 hours after a memory reactivation session conducted with or without lever access. The effects of DRN functional inactivation on long-term memory strength, as indicated by the magnitude of context-induced cocaine seeking, were assessed 72 hours later. Intra-DRN BM treatment immediately after memory reactivation with or without lever access attenuated subsequent context-induced cocaine-seeking behavior, independent of sex. Conversely, BM treatment in the adjacent periaqueductal gray (PAG) immediately after memory reactivation, or BM treatment in the DRN 6 hours after memory reactivation, did not alter responding. Together, these findings indicate that the DRN plays a requisite role in maintaining cocaine-memory strength during reconsolidation.

Keywords: memory reconsolidation, raphé, c-Fos, baclofen, muscimol, cocaine

1. INTRODUCTION

In the course of chronic drug use, initially neutral environmental stimuli become occasion setters, stimuli that predict access to drugs of abuse (Ehrman et al., 1992; O’Brien et al., 1992; Foltin and Haney, 2000; Bouton and Swartzentruber, 1986). Subsequent recall of the corresponding contextual drug memories elicits drug craving and relapse in individuals with substance use disorders (SUDs1; Kelley, 2004; Sorg, 2012), but it can also destabilize drug-associated memory traces and require reconsolidation into long-term memory stores to maintain their strength (Tronson and Taylor, 2007). Interference with memory reconsolidation can weaken drug-associated memories and stimulus control over drug-seeking behavior in rat models of drug relapse (Lee et al., 2006; Fuchs et al., 2009) and drug craving in individuals with SUDs (Saladin et al., 2013; Das et al., 2015; Yue et al., 2022). However, a more complete understanding of the neural mechanisms underlying memory reconsolidation is needed to inform the development of effective anti-relapse treatment strategies.

The raphé nuclei may contribute to memory reconsolidation based on their neurochemistry and anatomical connectivity. The raphé nuclei release several neurotransmitters and neuropeptides that have been implicated in memory reconsolidation, including glutamate (Milton et al., 2008; Lee and Everitt, 2008), dopamine (Yan et al., 2014), serotonin (Schmidt et al., 2017), and corticotrophin-releasing factor (Ritchie et al., 2021). The raphé nuclei also share monosynaptic connections with several brain regions that regulate cocaine-memory reconsolidation (Azmitia and Segal, 1978), including the medial prefrontal cortex (mPFC; Sorg et al., 2015), basolateral amygdala (BLA; Lee at al. 2005; Fuchs et al., 2009), and dorsal hippocampus (DH; Ramirez et al., 2009). The dorsal raphé nucleus (DRN) is of particular interest, as optogenetic or pharmacological disruption of DRN signaling interferes with fear memory consolidation (Sarihi et al., 2011; Sengupta and Holmes, 2019). However, the contributions of the DRN or other subdivisions of the midbrain raphé to memory reconsolidation have not been evaluated.

In the present study, we investigated the involvement of midbrain raphé nuclei in cocaine-memory reconsolidation. In the first experiment, we characterized subregion-specific neuronal activation in the raphé during memory reconsolidation compared to no memory reconsolidation by quantifying c-Fos expression, a marker of neuronal activation, in subregions of the DRN and in the median raphé nucleus (MRN). In subsequent experiments, we examined whether the functional integrity of the DRN in particular was necessary for cocaine-memory reconsolidation by transiently inhibiting the DRN using a cocktail of the GABAB/A agonists, baclofen plus muscimol (BM; 1.0 mM/0.1 mM, 0.5 uL), immediately after memory reactivation, when drug memories were expected to be labile, or six hours later, when drug memories were expected to be reconsolidated and thus resistant to manipulation. The memory-reactivation session was conducted with or without lever access to examine if lever access influenced memory destabilization or induced extinction learning. Lastly, BM treatment was administered into the periaqueductal gray (PAG) in a control experiment to determine whether observed effects were anatomically selective to the DRN. The PAG was selected because it is dorsally adjacent to the DRN; thus, it could be the target of unintended drug spread away from the DRN.

2. METHODS

2.1. Subjects

Male (N = 63; 275–325 g at the start of the experiment) and female (N = 60; 220–250 g at the start of the experiment) Sprague-Dawley rats were housed individually on a reversed light cycle (lights off at 6:00 a.m.). Each rat received ad libitum access to water and 20–25 g of standard rat chow per day. The rats were acclimated to handling prior to experimentation. The housing and care of animals was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and approved by the Washington State University Institutional Animal Care and Use Committee.

2.2. Food training

Rats received a 16-hour overnight food-training session to facilitate the acquisition of lever responding for un-signaled cocaine infusions subsequently. Food training took place in standard operant-conditioning chambers (26 × 27 × 27 cm; Coulbourn Instruments, Allentown, PA) housed within sound-attenuation cubicles (Med Associates, Fairfax, VT). Each chamber was equipped with two levers, a water bottle with sipper tube, a food tray, and a food-pellet dispenser controlled by Graphic State Notation software version 4.1.04 (Coulbourn). Water was available ad libitum. Responses on one lever (active lever) resulted in food reinforcement (45-mg grain-based pellets; Bio-Serv, Flemington, NJ) under a continuous reinforcement schedule. Responses on the other lever (inactive lever) had no programmed consequences. During the session, rats had no access to the distinct visual, olfactory, tactile, and auditory stimuli or to the chambers that were later paired with cocaine access. Rats received additional food training sessions, if needed, until they reached the acquisition criterion (≥ 100 active lever responses/session).

2.3. Surgery and catheter maintenance

Rats were anesthetized using ketamine and xylazine (100.0 mg/kg and 5.0 mg/kg, i.p., respectively). Back-mounted intravenous catheters were constructed in house and inserted into the right jugular vein as described previously (Fuchs et al., 2007). After the catheter surgery, the rats received unilateral guide cannula implants using standard stereotaxic procedures. A single, stainless-steel guide cannula (P1 Technologies, Roanoke, Virginia, USA) was directed towards the midline, 2 mm from the DRN (30° angle, AP −7.7 mm, ML +2.88 mm, DV −3.77 mm, relative to bregma) or the PAG (30° angle, AP −7.7 mm, ML +2.88 mm, DV −2.27 mm, relative to bregma). The cannula was angled to avoid damage to midline vasculature and cerebral aqueduct as previously described (Khodabande et al., 2021), and it was affixed to the skull using dental cement and jewelers’ screws. Rats received a bacon-flavored placebo tablet (5 g/tablet, p.o.; Bio-Serv) 24 hours prior to surgery to eliminate neophobia to the postoperative analgesic regimen. Postoperative analgesic treatment (bacon-flavored Rimadyl® MD tablets containing 2 mg carprofen per 5-g tablet; 5–8 mg/kg, p.o.; Bio-Serv) was provided for at least 48 hours after surgery. Rats received five days for post-surgical recovery. Catheters were flushed daily with an antibiotic solution of cefazolin (1.0 mg/0.1 mL; West-Ward, Eatontown, NJ; dissolved in 70 U/mL heparinized saline, Sagent Pharmaceuticals, Schaumburg, IL) followed by heparinized saline (0.1 mL, 10 U/mL) to help maintain catheter patency. The catheter and cannula were capped when not in use to prevent clogging (P1 Technologies). Catheter patency was assessed periodically using propofol (1 mg/0.1 mL, i.v.; Actavis Pharma, Parsippany, NJ), a short-acting anesthetic that produces rapid, temporary loss of muscle tone when administered intravenously.

2.4. Drug self-administration and extinction training

Drug self-administration training was conducted six days per week in operant-conditioning chambers (Coulbourn) configured to form two contexts. The first context consisted of a continuous red house light (0.4-fc brightness), intermittent pure tone (80 dB, 1 kHz; 2 sec on, 2 sec off), pine scented air freshener (Car Freshener Corp., Watertown, NY), and wire-mesh flooring (26 cm × 27 cm). The second context consisted of an intermittent white stimulus light over the inactive lever (1.2-fc brightness; 2 sec on, 2 secs off), continuous pure tone (75 dB, 2.5 kHz), vanilla-scented air freshener (Sopus Products, Moorpark, CA), and a slanted ceramic tile wall that bisected the bar flooring (19 cm × 27 cm). Rats were randomly assigned to context 1 or context 2 for cocaine self-administration training.

Drug self-administration training sessions took place during the rats’ dark cycle. During each session, active lever responses resulted in cocaine reinforcement (0.5 mg/kg per 50-μL infusion, delivered i.v. over 2 sec; NIDA Drug Supply Program, Research Triangle Park, NC) under a fixed ratio 1 schedule with a 20-second timeout period following each infusion. During the timeout period, active lever responses had no scheduled consequences. Inactive lever responses had no scheduled consequences at any time. Training continued for a minimum of 10 days or until rats reached an acquisition criterion of ≥10 infusions/session during at least ten 2-hour sessions. Rats then received daily 2-hour extinction training sessions in the alternate context. During the extinction training sessions, lever responses were recorded on both levers but had no scheduled consequences. The number of extinction sessions was set to seven to equalize memory age at the time of memory reactivation across rats. Immediately after extinction session 4, the rats were acclimated to the intracranial infusion procedure. A stainless-steel injection cannula (33-gauge; P1 Technologies) was inserted into the guide cannula to a depth of 2 mm past the tip of the guide cannula. It remained in place for 4 minutes with no infusion of fluids while the rats were held gently by the experimenter.

2.5. Memory reactivation

One day after the last extinction session, rats received a memory-reactivation (MR) session or remained in their home cages (no-reactivation controls; No-MR). Rats in the MR groups were placed into the cocaine-paired context and connected to the infusion lines for 15 min to elicit the retrieval and destabilization of contextual drug memories. Levers were either retracted or extended, but lever responses were not reinforced. Session length was selected based on parametric research indicating that 15-minute exposure to the cocaine-paired context with unreinforced lever access was sufficient to destabilize cocaine memories without producing overt behavioral extinction (Fuchs et al., 2009).

2.6. Experiment 1: Assessment of neuronal activation in midbrain raphé nuclei during cocaine-memory reconsolidation

We characterized neuronal activation, as indexed by c-Fos expression, in four midbrain raphé subregions in brain tissue collected 2 hours after the 15-minute memory-reactivation session or no memory reactivation (home cage stay). This time point was selected to capture peak c-Fos expression induced during the first 30 minutes of memory reconsolidation, based on the kinetics of c-Fos protein expression (i.e., expression approximately 90 minutes post induction; Zangenehpour and Chaudhuri, 2002). Group assignment was balanced based on mean active lever responding during the last three self-administration training sessions.

For tissue collection, rats were overdosed with ketamine hydrochloride and xylazine (100 and 5 mg/kg, i.v., or 300 and 15 mg/kg, i.p., respectively, depending on catheter patency) and were immediately perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde dissolved in PBS (PFA). The brains were post-fixed overnight in PFA at 4 °C and then placed into 30% sucrose/0.1% sodium azide solution until they sank. They were then rapidly frozen in powdered dry ice and stored at −80 °C. The brains were sliced into 30-μm coronal sections at −19 °C using a cryostat (Leica Biosystems, Buffalo Grove, IL). Sectioned brains were stored at 4 °C in 0.1% sodium azide dissolved in PBS.

Brain sections were washed in PBS and then incubated in blocking buffer containing 0.5% Triton X and 5% normal donkey serum (NDS) for 30 minutes. Brain sections were then incubated with mouse primary anti-c-Fos monoclonal IgG (sc-271243, Santa Cruz; 1:500) in blocking solution overnight at room temperature (RT). Next, the brain sections were washed in PBS (4 × 3 min) then incubated with donkey anti-mouse polyclonal IgG 488 (A21202, Thermo Fisher; 1:250) in PBS for 2 hours at RT. The sections were then stained with DAPI (D1306, Thermo Fisher; 1:1000) for 5 minutes and washed in PBS (4 × 3 min). The sections were mounted on glass slides and cover slipped using Prolong diamond antifade (P36965, Thermo Fisher). The edges of the cover slips were sealed with clear nail polish, and the slides were stored at 4 °C until imaging. Brain regions of interest (ROI) were identified using DAPI staining and the rat brain atlas (Paxinos and Watson, 2014). Images were acquired on an epifluorescence microscope (Leica CTR 6500, Wetzler, Germany) at 20x magnification. c-Fos-immunoreactive nuclei as well as ROI areas were quantified in ImageJ. Cell counts were converted to density values using ROI area (c-Fos/mm2) and averaged across duplicate images per subregion per subject.

2.7. Experiment 2: Effects of dorsal raphé neuronal inhibition immediately after memory reactivation on subsequent context-induced cocaine seeking

We next tested the hypothesis that DRN neuronal activity was necessary for cocaine-memory reconsolidation. Rats were randomly assigned to receive a memory reactivation session in the previously cocaine-paired context with the response levers extended (Lever groups) or retracted (No-Lever groups). Immediately after the memory-reactivation session, rats received a 0.5-μL, unilateral intra-DRN vehicle (PBS) or baclofen/muscimol (1.0 mM/0.1 mM, Alexis Biochemicals, dissolved in PBS) infusion, with treatment group assignment balanced based on the rats’ behavioral history as in experiment 1. The injection cannula was inserted into the guide cannula to a depth of 2 mm below the tip of the guide cannula. The intracranial infusion was administered over 2 minutes, with the injection cannula remaining in place for 1 min before and after infusion.

Twenty-four hours after the memory-reactivation session, daily extinction training sessions resumed in the designated extinction context. Lever responses during the first of these extinction sessions were analyzed to evaluate potential off-target effects of BM on extinction memory strength. Daily extinction training sessions continued until the rats reached an extinction criterion of ≤ 25 active lever responses/session on at least two consecutive days. Lever responses were then measured in the cocaine-paired context during a 2-hour test session, approximately three days after memory reactivation and intracranial treatment. Lever responses had no programmed consequences during the extinction training sessions and the test session.

2.8. Experiment 3: Effects of delayed dorsal raphé neuronal inhibition on subsequent context-induced cocaine seeking

We evaluated whether neuronal inactivation of the DRN would alter the strength of long-term cocaine memories. The experimental timeline was identical to that in experiment 2 except that rats were returned to their home cages after a memory-reactivation session with lever access and received intra-DRN vehicle or BM treatment 6 hours later, when memories were expected to be reconsolidated (Nader, 2015).

2.9. Experiment 4: Effect of periaqueductal gray neuronal inhibition immediately after memory reactivation on subsequent context-induced cocaine seeking

Lastly, we evaluated whether the effects of DRN inactivation observed in experiment 2 could be due to unintended BM spread along the cannula tract to the ipsilateral PAG. The experimental timeline was identical to that in experiment 2 except that the vehicle or BM infusion was administered unilaterally into the PAG immediately after a memory-reactivation session with lever access.

2.10. Brain histology

In experiments 2–4, Rats were overdosed with ketamine hydrochloride and xylazine (100 and 5 mg/kg, i.v., or 300 and 15 mg/kg, i.p., respectively, depending on catheter patency). The brains were extracted, rapidly frozen in methyl butane, and stored at −80 °C. The brains were sliced into 40-μm coronal sections at −19 °C on the cryostat. Brain sections were mounted on glass slides and stained with cresyl violet (Fisher Scientific, Waltham, MA). The most ventral portion of the cannula tract was identified using a light microscope.

2.11. Data analysis

Data from rats that failed to reach behavioral training criteria (n = 18) or had misplaced cannula (n = 36) were excluded from data analysis. Potential pre-existing group differences in cocaine infusions and/or lever responses during drug self-administration training, extinction training, and memory reactivation were assessed using mixed-factorial analyses of variance (ANOVA) with sex, subsequent group assignment (memory reactivation, MR; no-reactivation, No-MR), subsequent treatment (vehicle, BM), and subsequent lever access (lever, no-lever), as between-subjects factors, and time (training day) as within-subjects factor, where appropriate. c-Fos cell density (cells/mm2) was analyzed separately for each brain region using ANOVAs with reactivation (MR, No-MR) and sex as between-subjects factors. The total number of extinction sessions required to reach the extinction criterion, lever responses in the extinction and cocaine-paired contexts at test, the latency of the third active lever response in the cocaine-paired context at test (i.e., response latency; Fuchs et al., 2002) were assessed using ANOVAs with treatment, lever-access history, and sex, as the between-subjects factors, and context (extinction, cocaine-paired) and time (20-min bins), as within-subjects factors, where appropriate. Significant main and interaction effects were further probed using Tukey’s HSD or Sidak post hoc tests, where appropriate. Alpha was set at 0.05 for all analyses.

3. RESULTS

3.1. Experiment 1: Cocaine-memory reactivation induces c-Fos expression in the DRN during memory reconsolidation

The first experiment characterized c-Fos expression in three subregions of the DRN and in the MRN after memory reactivation, during reconsolidation (ns = 4–6 males/group, 5–6 females/group; Figure 1A). Full ANOVA results for behavioral history are reported in Table S1. Briefly, active (Figure 1B; 2 × 2 × 10 ANOVA; all Fs ≤ 3.14, ps ≥ 0.10) and inactive lever responding (2 × 2 × 10 ANOVA; all Fs ≤ 0.99, ps ≥ 0.40) were stable, while the number of cocaine infusions increased over time (2 × 2 × 10 ANOVA; time main effect only, F(9,134) = 28.81, p < 0.001; days 1–2 < days 3–10, Tukey’s tests, p < 0.05), independent of sex or subsequent group assignment during cocaine self-administration training. During extinction training, active lever responding decreased in all groups in the extinction context (2 × 2 × 7 ANOVA; time main effect only, F(6,96) = 30.29, p < 0.001; day 1 > days 2–7, Tukey’s tests, p < 0.05), while inactive lever responding varied as a function of subsequent group assignment depending on sex and training day (2 × 2 × 7 ANOVA; sex x group x time interaction, F(6,96) = 3.41, p = 0.004; day 1: male MR > male No-MR, day 1: female MR < female No-MR, male MR: day 1 > days 2–7, female No-MR: day 1 > days 3–7, Tukey’s tests, p < 0.05; time main effect, F(6,96) = 12.37, p < 0.001). Inactive lever responding was higher in the male MR group and lower in the female MR group compared to the respective No-MR groups on the first extinction day. Inactive lever responding then decreased in the male MR and female No-MR groups, eliminating group differences. Lastly, active and inactive lever responding did not vary by sex in the MR groups during the 15-minute memory reactivation session (Figure 1B; ts ≤ 0.36, ps ≥ 0.72).

Figure 1. Cocaine-memory reactivation elicits c-Fos expression in the dorsal raphé during memory reconsolidation.

Figure 1.

Open in a new tab

(A) Experimental timeline. Rats were trained to lever press for cocaine infusions in a distinct context, and their lever responding was extinguished in a second context. On post-cocaine day 8, the rats were re-exposed to the cocaine-paired context for 15 min to reactivate cocaine memories (memory reactivation; MR) or remained in their home cages (No-MR). Brain tissue was collected 2 hours later. (B) Cocaine infusions and lever responses (mean/2 hours ± SEM) during self-administration and extinction training and during the memory-reactivation session in the cocaine-paired context (mean/15 minutes ± SEM) by male (ns = 4–6 per group) and female (ns = 5–6 per group) rats. (C) Representative 5x and 20x images of midbrain raphé subregions of interest showing c-Fos-immunoreactive (IR; Red) and DAPI-stained (Blue) nuclei. Scale bars represent 50 μm. (D) c-Fos-IR cell density (mean number of IR nuclei per mm2 ± SEM) in tissue collected from male and female rats after MR or No-MR. Symbols: ‡/‡, ANOVA time main/simple effect; ±/+, ANOVA (subsequent) group main/simple effect. Abbreviations: Aq, cerebral aqueduct; dDRN, dorsal subregion of dorsal raphé; vDRN, ventral subregion of dorsal raphé; vlPAG, ventrolateral periaqueductal gray; lDRN, lateral subregion of dorsal raphé; VTg, ventral tegmental nucleus; MLF, medial longitudinal fasciculus; MRN, medial raphé.

After memory reactivation, c-Fos expression (Figure 1C–1D) increased in the dorsal DRN (dDRN; 2 × 2 ANOVA; group main effect only, F(1,16) = 18.53, p < 0.001; all other Fs ≤ 3.03, ps ≥ 0.10) and the ventral DRN (vDRN; 2 × 2 ANOVA; group main effect only, F(1,16) = 7.66, p = 0.01; all other Fs ≤ 0.04, ps ≥ 0.85) independent of sex during memory reconsolidation, compared to no memory reactivation (i.e., home cage stay). Interestingly, c-Fos expression increased in the lateral DRN (lDRN) in males, but not in females (2 × 2 ANOVA; sex × group interaction, F(1,16) = 4.82, p = 0.04; males: MR > No-MR, Sidak test, p < 0.05; group main effect, F(1,16) = 14.49, p = 0.002; sex main effect, F(1,16) = 0.04, p = 0.85) after memory reactivation. In contrast, c-Fos expression was not altered in the MRN in either sex (all Fs ≤ 1.67, p ≥ 0.22) after memory reactivation.

3.2. Experiment 2: DRN neuronal inhibition immediately after cocaine-memory reactivation reduces subsequent drug context-induced cocaine seeking

The second experiment evaluated whether the functional integrity of the DRN was necessary for maintaining labile cocaine memories and whether DRN engagement was dependent upon lever access (and potential extinction learning) during the memory reactivation session (Figure 2A). Male rats and female rats received a memory reactivation session either with the response levers extended (Lever) or retracted (No-Lever) followed immediately by intra-DRN vehicle or BM treatment (Figure 2B; ns = 6–7 males/group, 6–7 females/group). There were no pre-existing differences between the subsequent lever-access (not shown in Figure 2B–C) or subsequent treatment groups during cocaine self-administration and extinction training (see full ANOVA results for behavioral history in Table S1). Females exhibited more active lever responses (Figure 2C; 2 × 2 × 2 × 10 ANOVA; sex × time interaction, F(9,387) = 2.42, p < 0.01; day 1: females > males, females: day 1 > days 2–10, males: no day simple effects, Tukey’s tests, ps < 0.05) and obtained more cocaine infusions (sex × time interaction, F(9,387) = 2.02, p < 0.04; day 1: males < females, males: day 1 < days 6–10, females: day 1 > day 2 < days 7–10, Tukey tests, ps < 0.05) than males on the first cocaine self-administration day. In addition, the groups exhibited a decrease in inactive lever responding across cocaine self-administration training days (time main effect only, F(9,387) = 3.97, p < 0.001; day 1 > days 8–10, Tukey tests, ps < 0.05). During extinction training, all groups exhibited a decrease in active (Figure 2C; 2 × 2 × 2 × 7 ANOVA; time main effect only, F(6,264) = 138.58, p < 0.001) and inactive lever responding (time main effects only, F(6,264) = 26.87, p < 0.001) in the extinction context after the first day (day 1 > days 2–7, Tukey’s tests, ps < 0.05). The groups that received lever access did not differ in active (Figure 2D; 2 × 2 ANOVA; all Fs ≤ 0.09, ps ≥ 0.76) or inactive (all Fs ≤ 1.03, ps ≥ 0.32) lever responding during the memory reactivation session. Furthermore, the groups did not differ in the number of sessions required to reach the extinction criterion after the memory reactivation session as a function of sex, lever-access history, or intra-DRN BM treatment (2 × 2 × 2 ANOVA; all Fs ≤ 2.53, ps ≥ 0.12; mean ± SEM = 2.10 ± 0.06 days, not shown).

Figure 2. DRN neuronal inhibition during memory reconsolidation reduces cocaine-memory strength.

Figure 2.

Open in a new tab

(A) Experimental timeline. Rats were trained to lever press for cocaine infusions in a distinct context, and their lever responding was extinguished in a second context. On post-cocaine day 8, the rats were re-exposed to the cocaine-paired context for 15 min with or without lever access to elicit cocaine-memory reactivation (MR). Immediately after the MR session, the rats received an intra-DRN vehicle (VEH) or baclofen/muscimol (BM; 1.0/0.1 mM, 0.5 μL) infusion. Lever responding was then assessed in the extinction (EXT) and cocaine-paired contexts (COC). (B) Brain schematics adapted from Paxinos and Watson (2014) show injection cannula placements for subjects in the eight experimental groups. Numbers represent distance from Bregma in millimeters. (C) Cocaine infusions and lever responses (mean/2 hours ± SEM) in males (ns = 6–7 per group) and females (ns = 6–7 per group) during self-administration and extinction training sessions. (D) Active- and inactive lever responses during the memory-reactivation session (mean/15 minutes ± SEM) in males and females in the lever access groups. (E) Active- and inactive lever responses during the first re-exposure to the extinction and cocaine-paired contexts post treatment (mean/2 hours ± SEM). (F) Latency of the third active lever response (mean ± SEM) in the cocaine-paired context at test. (G) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test collapsed across sex. (H-I) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test in males and females, respectively. Symbols: ‡/‡, ANOVA time main/simple effect; */*ANOVA (subsequent) BM treatment main/simple effect; ⚥, ANOVA sex simple effect; #/#, ANOVA context main/simple main effect; §, ANOVA lever-access history simple effect; all ps < 0.05.

Upon re-exposure to the extinction and cocaine-paired contexts at test, intra-DRN BM treatment administered immediately after the memory reactivation session reduced active lever responding depending on the testing context, independent of sex or lever-access history (Figure 2E upper panel; 2 × 2 × 2 × 2 ANOVA; treatment x context interaction, F(1,44) = 21.59, p < 0.001; context main effect, F(1,44) = 235.16, p < 0.001; treatment main effect, F(1,44) = 23.78, p = < 0.001; all other Fs ≤ 1.06, ps ≥ 0.31). Collapsed across sex and lever-access history, exposure to the cocaine-paired context elicited more active lever responding in both treatment groups than exposure to the extinction context (Tukey’s tests, ps < 0.05). Furthermore, BM treatment reduced active lever responding in the cocaine-paired context (Tukey’s test, p < 0.05), but not in the extinction context, relative to vehicle treatment. Exposure to the cocaine-paired context resulted in more inactive lever responding than exposure to the extinction context, independent of sex, lever-access history, and treatment (Figure 2E, lower panel; 2 × 2 × 2 × 2 ANOVA; context main effect only, F(1,44) = 18.45, p < 0.001; all other Fs ≤ 2.29, ps ≥ 0.14). During the test session in the cocaine-paired context, BM treatment also increased the active-lever response latency compared to vehicle treatment, independent of sex or lever-access history (Figure 2F; 2 × 2 × 2 ANOVA; treatment main effect only, F(1,44) = 8.11, p = 0.007; all other Fs ≤ 1.29, ps ≥ 0.27). Lastly, the time-course analysis confirmed that BM treatment reduced active lever responding in the cocaine-paired context at test as a function of time, independent of sex or lever-access history (Figure 2G; 2 × 2 × 2 × 6 ANOVA; treatment x time interaction, F(5,220) = 3.68, p = 0.003; bins 1 and 3: BM < VEH; VEH: bin 1 > bins 2–6; BM: bin 1 > bins 2–6, Tukey’s tests, ps < 0.05; treatment main effect, F(1,46) = 19.45, p < 0.001; time main effect, F(5,220) = 64.67, p < 0.001; all other Fs ≤ 2.11, ps ≥ 0.07), compared to vehicle treatment. Separate ANOVAs for each sex revealed that lever-access history reduced active lever responding in males during the first 20 minutes of the test session, but intra-DRN BM treatment impaired active-lever responding independent of lever-access history (Figure 2H; 2 × 2 × 6 ANOVA; lever-access history x time interaction, F(5,110) = 2.32, p < 0.05; bin 1: No-Lever > Lever, No-Lever: bin 1 > 2–6, Lever: bin 1 > 3–6, Tukey’s tests, p ≤ 0.05; time main effect, F(5,110) = 30.79, p < 0.001; treatment main effect, F(1,22) = 14.43, p < 0.001; all other Fs ≤ 2.16, ps ≥ 0.06). Similarly, in females, BM treatment impaired active-lever responding independent of lever-access history or time, but active lever responding decreased across the test session independent of lever-access history or treatment (Figure 2I; 2 × 2 × 6 ANOVA; time main effect, F(5,110) = 34.31, p < 0.001; bin 1 > bins 2–6, Tukey’s tests, p < 0.05; treatment main effect, F(1,22) = 9.27, p = 0.006; all other Fs ≤ 2.01, ps ≥ 0.07).

3.3. Experiment 3: DRN neuronal inhibition 6 hours after cocaine-memory reactivation does not alter subsequent drug context-induced cocaine seeking

The third experiment assessed whether intra-DRN BM treatment (Figure 3A) altered memory strength after cocaine memories were reconsolidated into long-term memory stores. Rats received intra-DRN BM or vehicle treatment (Figure 3B) six hours after a memory reactivation session with lever access (ns = 7–8 males/group, 6–7 females/group). There were no pre-existing differences between the subsequent treatment groups during cocaine self-administration (see full ANOVA results for behavioral history in Table S1). Rats exhibited a time-dependent decrease in active (Figure 3C; 2 × 2 × 10 ANOVA; time main effect only, F(9,207) = 2.18, p = 0.03; Tukey’s Test, day 1 > day 4–7) and inactive (2 × 2 × 10 ANOVA; time main effect only, F(9,207) = 3.17, p < 0.001; day 1 > days 3–10, Tukey’s tests, p < 0.05) lever responding across the cocaine self-administration training days. The groups obtained more cocaine infusions on later training days, and males obtained more cocaine infusions overall than females (2 × 2 × 10 ANOVA; time main effect, F(1,207) = 10.07, p < 0.001, Tukey’s test, day 1 < days 5–10, p < 0.05; sex main effect, F(1,23) = 5.08, p = 0.03). However, there were no sex differences in the mean number of cocaine infusions obtained during the last three training days (2 × 2 ANOVA, all sex main and interaction effects, Fs < 0.27, p > 0.61). During the first day of extinction training, the female subsequent VEH treatment group responded less on the active lever than the respective male group (Figure 3C; 2 × 2 × 7 ANOVA; treatment × sex × time interaction, F(6,138) = 2.14, p = 0.05; day 1: male VEH > female VEH; all groups: day 1 > days 2–7, Tukey’s tests, ps < 0.05), after which active lever responding decreased in all groups. Inactive lever responding decreased across the extinction training days independent of sex or subsequent treatment (2 × 2 × 7 ANOVA; time main effect only, F(6,138) = 16.29, p < 0.001; day 1 > days 2–7, Tukey’s tests, ps < 0.05). The groups also did not differ in active (Figure 3D; 2 × 2 ANOVA; all Fs ≤ 2.85, all ps ≥ 0.11) or inactive (2 × 2 ANOVA; all Fs ≤ 1.36, all ps ≥ 0.26) lever responding during the memory reactivation session or in the number of extinction sessions required to reach the extinction criterion as a function of sex or delayed BM treatment (mean ± SEM = 2.00 ± 0.00 days, not shown).

Figure 3. DRN neuronal inhibition after reconsolidation does not alter cocaine-memory strength.

Figure 3.

Open in a new tab

(A) Experimental timeline. Rats were trained to self-administer cocaine in a distinct context, and their lever responding was extinguished in a second context. On post-cocaine day 8, rats were re-exposed to the cocaine-paired context for 15 min with lever access to elicit cocaine-memory reactivation (MR). Six hours later, the rats received an intra-DRN vehicle (VEH) or baclofen/muscimol (BM; 1.0/0.1 mM, 0.5 μL) infusion. Lever responding was then assessed in the extinction (EXT) and cocaine-paired contexts (COC). (B) Brain schematics adapted from Paxinos and Watson (2014) show injection cannula placements for subjects in the four experimental groups. Numbers represent distance from Bregma in millimeters. (C) Cocaine infusions and lever responses (mean/2 hours ± SEM) during self-administration and extinction training sessions in male (ns = 7 per group) and female (ns = 6–7 per group) rats. (D) Active- and inactive lever responses during the memory-reactivation session (mean/15 minutes ± SEM) in males and females. (E) Active- and inactive lever responses during the first re-exposure to the extinction and cocaine-paired contexts post treatment (mean/2 hours ± SEM). (F) Latency of the third active lever response (mean ± SEM) in the cocaine-paired context at test. (G) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test collapsed across sex. (H-I) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test in males and females, respectively. Symbols: ‡/‡, ANOVA time main/simple effect; ⚥/⚥, ANOVA sex main/simple effect; # ANOVA context main effect; all ps < 0.05.

Exposure to the cocaine-paired context at test elicited more active lever responding than exposure to the extinction context, independent of sex or delayed BM treatment (Figure 3E; 2 × 2 × 2 ANOVA; context main effect only, F(1,23) = 96.95, p < 0.001; all other Fs ≤ 0.45, ps ≥ 0.51). Similarly, exposure to the cocaine-paired context elicited more inactive lever responding than exposure to the extinction context, but inactive lever responding also varied by sex depending on treatment (2 × 2 × 2 ANOVA, sex x treatment interaction, F(1,23) = 5.28, p = 0.03; context main effect, F(1,23) = 6.66, p = 0.02; all other Fs ≤ 3.11, ps ≥ 0.09). Specifically, the female BM group responded less on the inactive lever than the male BM group, independent of testing context (Sidak’s test, p < 0.05). Neither delayed BM treatment nor sex altered active lever response latency in the cocaine-paired context at test (Figure 3F; 2 × 2 ANOVA; all Fs ≤ 0.52, ps ≥ 0.48). Similarly, the time-course analysis indicated that active lever responding in the cocaine-paired context decreased after the first 20 minutes of the test session independent of sex or delayed treatment (Figure 4G; 2 × 2 × 6 ANOVA; time main effect only, F(5,115) = 15.70, p < 0.001; bin 1 > bins 2–6, Tukey’s tests, ps < 0.05; all other Fs ≤ 1.60, ps ≥ 0.17). Similar effects were seen in separate analyses for males and females (Figures 4H–I; 2 × 6 ANOVAS; time main effects only, Fs(5,55,60) ≥ 5.25, ps < 0.001; males: bin 1 > bins 3–6, females: bin 1 > bins 2–6, Tukey’s tests, ps < 0.05).

Figure 4. PAG neuronal inhibition during memory reconsolidation does not alter cocaine-memory strength.

Figure 4.

Open in a new tab

(A) Experimental timeline. Rats were trained to self-administer cocaine in a distinct context, and their lever responding was extinguished in a second context. On post-cocaine day 8, rats were re-exposed to the cocaine-paired context for 15 min with access to unreinforced levers to elicit memory reactivation (MR). Immediately after the session, the rats received an intra-PAG vehicle (VEH) or baclofen/muscimol (BM; 1.0/0.1 mM, 0.5 uL) infusion. Lever responding was then assessed in the extinction (EXT) and cocaine-paired contexts (COC). (B) Brain schematics adapted from Paxinos and Watson (2014) show injection cannula placements for subjects in the four experimental groups. Numbers represent distance from Bregma in millimeters. (C) Cocaine infusions and lever responses (mean/2 hours ± SEM) during self-administration and extinction training sessions in male (ns = 6 per group) and female (ns = 5 per group) rats. (D) Active- and inactive lever responses during the memory-reactivation session (mean/15 minutes ± SEM) in males and females in the lever access groups. (E) Active- and inactive lever responses during the first re-exposure to the extinction and cocaine-paired contexts post treatment (mean/2 hours ± SEM). (F) Latency of the third active lever response (mean ± SEM) in the cocaine-paired context at test. (G) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test collapsed across sex. (H-I) Time-course of active lever responding (mean ± SEM) in the cocaine-paired context at test in males and females, respectively. Symbols: ‡/‡, ANOVA time main/simple effect; ⚥, ANOVA sex simple effect; *, ANOVA subsequent BM treatment simple effect; #, ANOVA context main effect; all ps < 0.05.

3.4. Experiment 4: PAG neuronal inhibition immediately after memory reactivation does not alter subsequent context-induced cocaine seeking

The fourth experiment investigated whether the treatment effects observed in experiment 2 could be produced by BM diffusion from the DRN into the neighboring PAG during cocaine-memory reconsolidation (Figure 4A). Male rats and female rats received BM or vehicle infusions into the PAG immediately after a memory reactivation session with lever access (Figure 4B; ns = 7 males/group, 5 females/group). There were no pre-existing differences between the subsequent treatment groups during cocaine self-administration training (see full ANOVA results for behavioral history in Table S1). Briefly, all groups exhibited stable active (Figure 4C; 2 × 2 × 10 ANOVA; all Fs ≤ 1.77, ps ≥ 0.08) and inactive (2 × 2 × 10 ANOVA; all Fs ≤ 2.15, ps ≥ 0.08) lever responding and a gradual increase in the number of cocaine infusions (2 × 2 × 10 ANOVA; time main effect only, F(9,180) = 5.67, p < 0.001, Tukey’s tests, day 3 < days 5–10, ps < 0.05) across the cocaine self-administration training days. On the first and second days of extinction training, females exhibited more active lever responding in the extinction context than males, and active lever responding gradually decreased in both sexes thereafter (2 × 2 × 7 ANOVA; sex x time interaction, F(6,120) = 5.01, p = 0.001; days 1–2: females > males, males: day 1 > days 2–7; females: day 1 > day 2 > days 3–7, Tukey’s tests, ps < 0.05; time main effect, F(6,120) = 36.60, p < 0.001). The subsequent VEH groups exhibited more inactive lever responding than the subsequent BM treatment groups on the first extinction training day, independent of sex (2 × 2 × 7 ANOVA; subsequent treatment x time interaction, F(6,120) = 3.95, p = 0.001; day 1: VEH > BM; VEH: day 1 > day 2–7, Tukey’s tests, ps < 0.05; time main effect, F(6,120) = 10.94, p < 0.001). The groups did not differ in active (Figure 4D; 2 × 2 ANOVA; all Fs ≤ 3.04, ps ≥ 0.10) and inactive (2 × 2 ANOVA; all Fs ≤ 0.89, ps ≥ 0.36) lever responding during the memory reactivation session. Furthermore, the groups did not differ in the number of sessions required to reach the extinction criterion as a function of sex or intra-PAG BM treatment (2 × 2 × 2 ANOVA; all Fs ≤ 0.69, ps ≥ 0.41; mean ± SEM = 2.04 ± 0.04 days, not shown).

Exposure to the cocaine-paired context increased active (Figure 4E; 2 × 2 × 2 ANOVA; context main effect only, F(1,20) = 39.53, p < 0.001; all other Fs ≤ 0.96, ps ≥ 0.34) and inactive (2 × 2 × 2 ANOVA; context main effect only, F(1,20) = 6.39, p < 0.02; all other Fs ≤ 4.19, ps ≥ 0.054) lever responding, independent of sex or intra-PAG treatment, compared to exposure to the extinction context. Thus, intra-PAG BM treatment administered immediately after memory reactivation did not alter active- or inactive lever responding in either context at test. Similarly, neither sex nor BM treatment altered active lever response latency in the cocaine-paired context at test (Figure 4F; 2 × 2 ANOVA, all Fs ≤ 0.57, ps ≥ 0.46). The time-course analysis indicated that active lever responding in the cocaine-paired context decreased after the first 20 minutes of the test session, independent of sex or treatment (Figure 4G; 2 × 2 × 6 ANOVA; time main effect only, F(5,100) = 10.67, p < 0.001; bin 1 > bins 3–6, Tukey’s tests, ps < 0.05; all other Fs ≤ 1.58, ps ≥ 0.17). Similar effects were seen in separate analyses for males and females (Figures 4H–I; 2 × 6 ANOVAS; time main effects only, Fs(5,40–60) ≥ 2.40, ps ≤ 0.05, males: bin 1 > 3–6, females: bin 1 > 6, Tukey’s tests, ps < 0.05).

4. Discussion

The primary finding of this study is that the DRN plays a requisite role in cocaine-memory reconsolidation. To demonstrate this, we first mapped subregions of the midbrain raphé that exhibited neuronal activation, as indicated by c-Fos expression, during the putative time of cocaine-memory reconsolidation in experiment 1. We discovered such increases in c-Fos expression in the dorsal, ventral, and lateral subregions of the DRN, but not in the MRN (Figure 1). Next, we evaluated whether DRN neuronal activity was necessary for cocaine-memory maintenance in experiments 2–4. We observed that GABAA/B agonist-induced DRN inhibition immediately after cocaine-memory reactivation increased the latency and reduced the magnitude of drug-seeking behavior in the cocaine-context 72 hours post treatment, independent of sex or lever access during the preceding memory-reactivation session (Figure 2). Conversely, DRN inhibition 6-hours after cocaine-memory reactivation (Figure 3) or PAG inhibition immediately after cocaine-memory reactivation (Figure 4) did not reduce subsequent cocaine-seeking behavior or increase response latency. Thus, intra-DRN BM treatment reduced cocaine seeking in a context-specific, memory reactivation-dependent, and anatomically selective manner independent of sex. Therefore, we postulate that the functional integrity of the DRN is necessary for the maintenance of cocaine-memory strength during reconsolidation. These findings extend the list of brain regions involved in cocaine-memory reconsolidation and provide impetus for future research inquiries into the cellular and circuit mechanisms by which the DRN regulates the strength of contextual cocaine memories that facilitate drug craving and relapse.

Lever access during the memory-reactivation session did not alter response latency (Figure 2F) or the overall magnitude of cocaine-seeking behavior during the test session (Figure 2E) in the control groups, relative to no-lever access. We experimentally manipulated lever access during the memory reactivation session to alter the extent of instrumental extinction learning and thus possible treatment effects on extinction-memory consolidation (Figure 2). Drug-memory reconsolidation and extinction-memory consolidation are pharmacologically distinct processes (Suzuki et al., 2004; Tronson and Taylor, 2007; Bustos et al., 2009) that co-occur following a non-reinforced memory reactivation session according to the trace dominance hypothesis (Eisenberg et al., 2003). These processes stabilize context-response-drug and context-response-NO-drug (extinction) associative memories, respectively, and these memories compete subsequently to determine the magnitude of context-induced cocaine-seeking behavior (Eisenberg et al., 2003). It is likely that lever access during the 15-minute memory reactivation session results in the formation of weak extinction memories that are insufficient to suppress subsequent cocaine-seeking behavior or produce overt behavioral extinction (Fuchs et al. 2009). Theoretically, such weak extinction memories could increase in strength or salience in response to experimental manipulations that enhance their consolidation. In our study, lever access during the memory-reactivation session did attenuate active lever responding in males, but not in females, during the first 20-minutes of the subsequent test session, compared to no-lever access (Figures 2H–I). However, BM treatment in the DRN diminished cocaine-seeking behavior in both sexes independent of lever-access history (Figures 2E, 2G), supporting the interpretation that DRN inhibition genuinely impaired cocaine-memory strength independent of potential effects on extinction-memory strength.

Research examining the contributions of midbrain raphé nuclei to memory function has been limited. Here, we focused on the role of the DRN in memory reconsolidation, but previous studies demonstrated that both the DRN and the MRN are involved in memory consolidation in several paradigms (Sarihi et al., 1999; Wang et al., 2015). DRN neuronal activation is critical for fear-memory consolidation, as indicated by deficits in Pavlovian fear responses after post-training administration of the sodium channel blocker, tetrodotoxin, into this brain region (Sarihi et al., 2011). Furthermore, arginine-vasopressin signaling in the DRN is necessary and sufficient for passive-avoidance memory consolidation (Kovács et al., 1979; Kovács et al., 1980), and orexin signaling in the DRN is required for spatial reference-memory consolidation (Khodabande et al., 2021). This literature and the present findings suggest that the DRN plays a fundamental role in memory storage, regulating both consolidation and reconsolidation. While the MRN is necessary for fear-memory consolidation (Wang et al., 2015), we did not investigate its functional role in memory reconsolidation due to negative c-Fos findings (Figure 1D). Notably, the null effect of memory reactivation on c-Fos expression in the MRN must be interpreted with caution as c-Fos expression provides only a snapshot of neuronal activation (Labiner et al., 1993; Cullinan et al., 1995; Zangenehpour and Chaudhuri, 2002), potentially failing to capture MRN engagement (i.e., increase or decrease in neuronal activation) at other time points during memory reconsolidation or through intracellular signaling mechanisms that do not evoke significant changes in c-Fos expression.

The DRN likely regulates the strength of reconsolidating cocaine memories as a component of a larger neural circuitry. It receives diverse inputs via glutamatergic and non-glutamatergic fibers from the mPFC, lateral habenula, and hypothalamus (Lee et al., 2003; Soiza-Reilly and Commons, 2011); GABAergic fibers from the VTA, substantia nigra, rostral tegmental nucleus, and PAG (Soiza-Reilly et al., 2013; Kirouac and Mabrouk, 2004); adrenergic fibers from the locus coeruleus (Kim et al., 2004); and cholinergic fibers from the thalamus (Hernandez-Lopez et al., 2013). Glutamatergic and GABAergic fibers terminate in close proximity within the DRN, frequently forming axo-axonic synapses and functional triads with postsynaptic DRN neurons (Soiza-Reilly et al., 2013). Putative interactions between these glutamate and GABA terminals and local GABAergic interneurons likely regulate DRN excitability with precision during memory reconsolidation. In turn, DRN efferents terminate in several brain regions, including the mPFC, nucleus accumbens, BLA, DH, and locus coeruleus (Azmitia and Segal, 1978; Kim et al., 2004) that are critical to cocaine-memory reconsolidation (Lee et al., 2005; Miller and Marshal, 2005; Sorg et al., 2012; Qi et al., 2022), as well as to the hypothalamus, VTA, and substantia nigra (Hernandeź-López et al., 2013). Future research will aim to dissect the role of specific DRN efferent and afferent pathways in cocaine-memory reconsolidation.

Transient sex differences in cocaine intake varied across our experiments (Figures 2C, 3C), as across previous studies (Lynch and Carroll, 1999; Caine et al. 2004; Fuchs et al. 2005), but did not impact cocaine-memory strength as indicated by the magnitude of cocaine-seeking behavior in the control groups at test (Figures 2E, 3E). Furthermore, DRN neuronal inhibition disrupted cocaine-memory strength independent of sex (Figure 2G) despite that c-Fos expression significantly increased in the lDRN of males, but not females, after cocaine-memory reactivation (Figure 1D). A small and inconsistent literature suggests the existence of both sex-dependent and sex-independent mechanisms for memory reconsolidation. For instance, systemic cortisol treatment enhances fear-memory reconsolidation in men, but not in women (Drexler et al., 2016); while stimulation of corticotropin-releasing factor signaling in the BLA enhances cocaine-memory reconsolidation in female, but not male, rats (Ritchie et al., 2021). Yet other reports indicate that some manipulations have sex-independent effects on memory reconsolidation. For example, systemic α2-adrenergic agonist and intra-hippocampal calpain inhibitor treatments impair auditory and contextual fear memory reconsolidation, respectively, in both sexes (Gamache et al., 2012; Nagayoshi et al., 2017). We predicted to see sex differences in DRN engagement in memory reconsolidation based on reports indicating sex differences in DRN anatomy and physiology, including sex differences in DRN efferent fiber density and DRN neuronal responses to stress and cocaine administration, respectively (Westenbroek et al., 2003; Goel et al., 2014; Perez et al., 2018; Petersen et al., 2020). GABA agonist-induced DRN neuronal inhibition is a blunt tool that might fail to detect distinct sex-specific mechanisms in the DRN that facilitate memory maintenance. In addition, some experimental parameters (e.g., absence of a stressor or acute cocaine exposure) in the present study might preclude sex-dependent DRN engagement in cocaine-memory reconsolidation. Inconsistencies in this limited literature underscore the importance of including sex as a biological variable in future studies.

5. Conclusion

Our findings indicate that the DRN is a critical locus for maintaining the strength of labile cocaine memories and, thus, contextual control over cocaine-seeking behavior in a rat model of drug relapse. The DRN is in an ideal position to regulate the strength of reward-associated memories based on its involvement in appetitive associative learning and in the encoding of anticipated reward value (Nakamura, 2013; Luo et al., 2015). In support of the latter, DRN neurons exhibit changes in firing rate that track the presentation or omission of reward-predictive stimuli (Nakamura et al., 2008; Liu et al., 2014), and optogenetic stimulation of these neurons has both rewarding and reinforcing effects (Liu et al., 2014; Nagai et al., 2020). We suggest that DRN neurons convey information about the affective salience and motivational significance of the cocaine-paired context during the memory reactivation session, and this information is reincorporated into the reconsolidating memory trace. Furthermore, chronic cocaine exposure, stress, and other factors may produce alterations in DRN cell and circuit function, giving rise to unusually strong or intrusive drug memories.

Supplementary Material

1

Highlights.

  • Labile associative memories are maintained via a reconsolidation process

  • DRN exhibits c-Fos expression during memory reconsolidation

  • DRN inhibition during reconsolidation reduces cocaine-memory strength

Acknowledgements

The authors are grateful to David Soto for expert technical assistance. This work was supported by NIH NIDA 2 R01 DA025646 (RAF), NIH NIDA 1 R01 DA057330 (RAF), Washington State Initiative 171 research funds administered through the WSU Alcohol and Drug Abuse Research program (JLR), and the Poncin Foundation Research Fund (JLR).

Footnotes

1

Abbreviations: basolateral amygdala, BLA; baclofen/muscimol, BM; dorsal raphé nucleus, DRN; dorsal raphé nucleus dorsal subregion, dDRN; dorsal raphé nucleus lateral subregion, lDRN; medial prefrontal cortex, mPFC; median raphé nucleus, MRN; periaqueductal gray, PAG; substance use disorder, SUD; dorsal raphé nucleus ventral subregion, vDRN

Declarations of interest: none

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Azmitia EC, Segal M, 1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J Comp Neurol 179, 641–668. DOI: 10.1002/cne.901790311. [DOI] [PubMed] [Google Scholar]
  2. Bouton ME, & Swartzentruber D (1986). Analysis of the associative and occasion-setting properties of contexts participating in Pavlovian discrimination. J Exp Psychol Anim Behav Process, 12(4), 333–350. DOI: 10.1037/0097-7403.12.4.333. [DOI] [Google Scholar]
  3. Bustos SG, Maldonado H, Molina VA, 2009. Disruptive effect of midazolam on fear memory reconsolidation: decisive influence of reactivation time span and memory age. Neuropsychopharmacology 34, 446–457. DOI: 10.1038/npp.2008.75. [DOI] [PubMed] [Google Scholar]
  4. Caine SB, Bowen CA, Yu G, Zuzga D, Negus SS, & Mello NK (2004). Effect of gonadectomy and gonadal hormone replacement on cocaine self-administration in female and male rats. Neuropsychopharmacology, 29(5), 929–942. doi: 10.1038/sj.npp.1300387 [DOI] [PubMed] [Google Scholar]
  5. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ, 1995. Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64, 477–505. DOI: 10.1016/0306-4522(94)00355-9. [DOI] [PubMed] [Google Scholar]
  6. Das RK, Lawn W, Kamboj SK, 2015. Rewriting the valuation and salience of alcohol-related stimuli via memory reconsolidation. Transl Psychiatry 5, 1–9. DOI: 10.1038/tp.2015.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ehrman RN, Robbins SJ, Childress AR, O’Brien CP, 1992. Conditioned responses to cocaine-related stimuli in cocaine abuse patients. Psychopharmacology (Berl) 107, 523–529. DOI: 10.1007/BF02245266. [DOI] [PubMed] [Google Scholar]
  8. Eisenberg M, Kobilo T, Berman DE, Dudai Y, 2003. Stability of retrieved memory: Inverse correlation with trace dominance. Science 301, 1102–1104. DOI: 10.1126/science.1086881. [DOI] [PubMed] [Google Scholar]
  9. Foltin RW, Haney M, 2000. Conditioned effects of environmental stimuli paired with smoked cocaine in humans. Psychopharmacology (Berl) 149, 24–33. DOI: 10.1007/s002139900340. [DOI] [PubMed] [Google Scholar]
  10. Fuchs RA, Bell GH, Ramirez DR, Eaddy JL, Su ZI, 2009. Basolateral amygdala involvement in memory reconsolidation processes that facilitate drug context-induced cocaine seeking. Eur J Neurosci 30, 889–900. DOI: 10.1111/j.1460-9568.2009.06888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuchs RA, Eaddy JL, Su ZI, Bell GH, 2007. Interactions of the basolateral amygdala with the dorsal hippocampus and dorsomedial prefrontal cortex regulate drug context-induced reinstatement of cocaine-seeking in rats. Eur J Neurosci 26, 487–498. DOI: 10.1111/j.1460-9568.2007.05674.x. [DOI] [PubMed] [Google Scholar]
  12. Fuchs RA, Evans KA, Mehta RH, Case JM, & See RE (2005). Influence of sex and estrous cyclicity on conditioned cue-induced reinstatement of cocaine-seeking behavior in rats. Psychopharmacology (Berl), 179(3), 662–672. doi: 10.1007/s00213-004-2080-7 [DOI] [PubMed] [Google Scholar]
  13. Fuchs RA, Weber SM, Rice HJ, Neisewander JL., 2002. Effects of excitotoxic lesions of the basolateral amygdala on cocaine-seeking behavior and cocaine conditioned place preference in rats. Brain Res 929, 15–25. DOI: 10.1016/s0006-8993(01)03366-2. [DOI] [PubMed] [Google Scholar]
  14. Gamache K, Pitman RK, Nader K, 2012. Preclinical evaluation of reconsolidation blockade by clonidine as a potential novel treatment for posttraumatic stress disorder. Neuropsychopharmacology 37, 2789–2796. DOI: 10.1038/npp.2012.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goel N, Innala L, Viau V, 2014. Sex differences in serotonin (5-HT) 1A receptor regulation of HPA axis and dorsal raphe responses to acute restraint. Psychoneuroendocrinology 40, 232–241. DOI: 10.1016/j.psyneuen.2013.11.020. [DOI] [PubMed] [Google Scholar]
  16. Hernandez-Lopez S, Garduño J, Mihailescu S (2013). Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus. Rev Neurosci, 24(5), 455–469. DOI: 10.1515/revneuro-2013-0012. [DOI] [PubMed] [Google Scholar]
  17. Kelley AE, 2004. Memory and addiction: shared neural circuitry and molecular mechanisms. Neuron 44, 161–179. DOI: 10.1016/j.neuron.2004.09.016. [DOI] [PubMed] [Google Scholar]
  18. Khodabande F, Akbari E, Ardeshiri MR, 2021. The modulation of the spatial reference memory by the orexinergic system of the dorsal raphe nucleus. Life Sci 265, 118777. DOI: 10.1016/j.lfs.2020.118777. [DOI] [PubMed] [Google Scholar]
  19. Kim MA, Lee HS, Lee BY, Waterhouse BD, 2004. Reciprocal connections between subdivisions of the dorsal raphe and the nuclear core of the locus coeruleus in the rat. Brain Res 1026, 56–67. DOI: 10.1016/j.brainres.2004.08.022. [DOI] [PubMed] [Google Scholar]
  20. Kirouac GJ, Li S, Mabrouk G, 2004. GABAergic projection from the ventral tegmental area and substantia nigra to the periaqueductal gray region and the dorsal raphe nucleus. J Comp Neurol 469, 170–184. DOI: 10.1002/cne.11005. [DOI] [PubMed] [Google Scholar]
  21. Kovacs GL, Bohus B, Versteeg DHG, Kloet ER, Wied D, 1979. Effect of oxytocin and vasopressin on memory consolidation: Sites of action and catecholaminergic correlates after local microinjection into limbic-midbrain structures. Brain Res 175, 303–314. DOI: 10.1016/0006-8993(79)91009-6. [DOI] [PubMed] [Google Scholar]
  22. Kovacs GL, Vecsei L, Medve L, Telegdy G, 1980. Effect on memory processes of anti-vasopressin serum microinjected into the dorsal raphe nucleus: the role of catecholaminergic neurotransmission. Exp Brain Res 38, 357–361. DOI: 10.1007/bf00236656. [DOI] [PubMed] [Google Scholar]
  23. Labiner DM, Butler LS, Cao Z, Hosford DA, Shin C, McNamara JO, 1993. Induction of c-fos mRNA by kindled seizures: complex relationship with neuronal burst firing. J Neurosci 13, 744–751. DOI: 10.1523/JNEUROSCI.13-02-00744.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee HS, Kim MA, Valentino RJ, Waterhouse BD, 2003. Glutamatergic afferent projections to the dorsal raphe nucleus of the rat. Brain Res 963, 57–71. DOI: 10.1016/s0006-8993(02)03841-6. [DOI] [PubMed] [Google Scholar]
  25. Lee JL, Di Ciano P, Thomas KL, Everitt BJ., 2005. Disrupting reconsolidation of drug memories reduces cocaine-seeking behavior. Neuron. 47, 795–801. DOI: 10.1016/j.neuron.2005.08.007. [DOI] [PubMed] [Google Scholar]
  26. Lee JL, Everitt BJ, 2008. Appetitive memory reconsolidation depends upon NMDA receptor-mediated neurotransmission. Neurobiol Learn Mem 90, 147–154. DOI: 10.1016/j.nlm.2008.02.004. [DOI] [PubMed] [Google Scholar]
  27. Lee JL, Milton AL, Everitt BJ, 2006. Reconsolidation and extinction of conditioned fear: inhibition and potentiation. J Neurosci 26, 10051–10056. DOI: 10.1523/JNEUROSCI.2466-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu Z, Zhou J, Li Y, Hu F, Lu Y, Ma M, Feng Q, Zhang JE, Wang D, Zeng J, Bao J, Kim JY, Chen ZF, El Mestikawy S, Luo M (2014). Dorsal raphe neurons signal reward through 5-HT and glutamate. Neuron, 81(6), 1360–1374. DOI: 10.1016/j.neuron.2014.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Luo M, Zhou J, Liu Z, 2015. Reward processing by the dorsal raphe nucleus: 5-HT and beyond. Learn Mem 22, 452–460. DOI: 10.1101/lm.037317.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lynch WJ, Carroll ME (1999). Sex differences in the acquisition of intravenously self-administered cocaine and heroin in rats. Psychopharmacology (Berl), 144, 77–82. doi: 10.1007/s002130050979 [DOI] [PubMed] [Google Scholar]
  31. Miller CA, & Marshall JF (2005). Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory. Neuron, 47(6), 873–884. DOI: 10.1016/j.neuron.2005.08.006. [DOI] [PubMed] [Google Scholar]
  32. Milton AL, Lee JL, Butler VJ, Gardner R, Everitt BJ, 2008. Intra-amygdala and systemic antagonism of NMDA receptors prevents the reconsolidation of drug-associated memory and impairs subsequently both novel and previously acquired drug-seeking behaviors. J Neurosci 28, 8230–8237. DOI: 10.1523/JNEUROSCI.1723-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nader K (2015). Reconsolidation and the Dynamic Nature of Memory. Cold Spring Harb Perspect Biol, 7(10), a021782. DOI: 10.1101/cshperspect.a021782Fa. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nagayoshi T, Isoda K, Mamiya N, Kida S, 2017. Hippocampal calpain is required for the consolidation and reconsolidation but not extinction of contextual fear memory. Mol Brain 10, 61. DOI: 10.1186/s13041-017-0341-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakamura K, 2013. The role of the dorsal raphe nucleus in reward-seeking behavior. Front Integr Neurosci 7, 60. DOI: 10.3389/fnint.2013.00060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nakamura K, Matsumoto M, Hikosaka O (2008). Reward-dependent modulation of neuronal activity in the primate dorsal raphe nucleus. J Neurosci, 28(20), 5331–5343. DOI: 10.1523/JNEUROSCI.0021-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nagai Y, Takayama K, Nishitani N, Andoh C, Koda M, Shirakawa H, Nakagawa T, Nagayasu K, Yamanaka A, Kaneko S (2020). The role of dorsal raphe serotonin neurons in the balance between reward and aversion. Int J Mol Sci, 21(6). DOI: 10.3390/ijms21062160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O’brien CP, Childress AR, Mclellan AT, Ehrman R, 1992. Classical conditioning in drug-dependent humans. Ann N Y Acad Sci 654, 400–415. DOI: 10.1111/j.1749-6632.1992.tb25984.x. [DOI] [PubMed] [Google Scholar]
  39. Paxinos G, Watson C, 2014. The Rat Brain in Stereotaxic Coordinates 7th Edition. Academic Press. ISBN: 9780123919496. [Google Scholar]
  40. Perez PD, Hall G, Zubcevic J, Febo M (2018). Cocaine differentially affects synaptic activity in memory and midbrain areas of female and male rats: an in vivo MEMRI study. Brain Imaging Behav, 12(1), 201–216. DOI: 10.1007/s11682-017-9691-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Petersen CL, Koo A, Patel B, Hurley LM, 2020. Serotonergic innervation of the auditory midbrain: dorsal raphe subregions differentially project to the auditory midbrain in male and female mice. Brain Struct Funct 225, 1855–1871. DOI: 10.1007/s00429-020-02098-3. [DOI] [PubMed] [Google Scholar]
  42. Qi S, Tan SM, Wang R, Higginbotham JA, Ritchie JL, Ibarra CK, Arguello AA, Christian RJ, Fuchs RA, 2022. Optogenetic inhibition of the dorsal hippocampus CA3 region during early-stage cocaine-memory reconsolidation disrupts subsequent context-induced cocaine seeking in rats. Neuropsychopharmacology 47, 1473–1483. DOI: 10.1038/s41386-022-01342-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ramirez DR, Bell GH, Lasseter HC, Xie X, Traina SA, Fuchs RA (2009). Dorsal hippocampal regulation of memory reconsolidation processes that facilitate drug context-induced cocaine-seeking behavior in rats. Eur J Neurosci, 30(5), 901–912. DOI: 10.1111/j.1460-9568.2009.06889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ritchie JL, Walters JL, Galliou JMC, Christian RJ, Qi S, Savenkova MI, Ibarra CK, Grogan SR, Fuchs RA, 2021. Basolateral amygdala corticotropin-releasing factor receptor type 1 regulates context-cocaine memory strength during reconsolidation in a sex-dependent manner. Neuropharmacology, 108819. DOI: 10.1016/j.neuropharm.2021.108819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Saladin ME, Gray KM, McRae-Clark AL, Larowe SD, Yeatts SD, Baker NL, Hartwell KJ, Brady KT, 2013. A double blind, placebo-controlled study of the effects of post-retrieval propranolol on reconsolidation of memory for craving and cue reactivity in cocaine dependent humans. Psychopharmacology (Berl) 226, 721–737. DOI: 10.1007/s00213-013-3039-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sarihi A, Yazdia M, Heshmatian B, Salehia I, Behzadi G, Naghdi N, Shahidi S, Komaki A, Haghparast A, Emam AH, 2011. The effects of lidocaine reversible inactivation of the dorsal raphe nucleus on passive avoidance learning in rats. Basic Clin Neurosci 2, 27–35. ISSN: 2228–7442 (Electronic); 2008–126X (Print) [Google Scholar]
  47. Sarihi A, Motamedi F, Rashidy-Pour A, Naghdi N, Behzadi G, 1999. Reversible inactivation of the median raphe nucleus enhances consolidation and retrieval but not acquisition of passive avoidance learning in rats. Brain Res 817, 59–66. DOI: 10.1016/S0006-8993(98)01196-2. [DOI] [PubMed] [Google Scholar]
  48. Schmidt SD, Furini CRG, Zinn CG, Cavalcante LE, Ferreira FF, Behling JAK, Myskiw JC, Izquierdo I, 2017. Modulation of the consolidation and reconsolidation of fear memory by three different serotonin receptors in hippocampus. Neurobiol Learn Mem 142, 48–54. DOI: 10.1016/j.nlm.2016.12.017. [DOI] [PubMed] [Google Scholar]
  49. Sengupta A, Holmes A, 2019. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory. Neuron 103, 489–505 e487. DOI: 10.1016/j.neuron.2019.05.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Soiza-Reilly M, Anderson WB, Vaughan CW, Commons KG, 2013. Presynaptic gating of excitation in the dorsal raphe nucleus by GABA. Proc Natl Acad Sci U S A 110, 15800–15805. DOI: 10.1073/pnas.1304505110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Soiza-Reilly M, Commons KG, 2011. Glutamatergic drive of the dorsal raphe nucleus. J Chem Neuroanat 41, 247–255. DOI: 10.1016/j.jchemneu.2011.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sorg BA, 2012. Reconsolidation of drug memories. Neurosci Biobehav Rev 36, 1400–1417. DOI: 10.1016/j.neubiorev.2012.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sorg BA, Todd RP, Slaker M, Churchill L, 2015. Anisomycin in the medial prefrontal cortex reduces reconsolidation of cocaine-associated memories in the rat self-administration model. Neuropharmacology 92, 25–33. DOI: 10.1016/j.neuropharm.2014.12.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Suzuki A, Josselyn SA, Frankland PW, Masushige S, Silva AJ, Kida S, 2004. Memory reconsolidation and extinction have distinct temporal and biochemical signatures. J Neurosci 24, 4787–4795. DOI: 10.1523/JNEUROSCI.5491-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tronson NC, Taylor JR, 2007. Molecular mechanisms of memory reconsolidation. Nat Rev Neurosci 8, 262–275. DOI: 10.1038/nrn2090. [DOI] [PubMed] [Google Scholar]
  56. Wang DV, Yau HJ, Broker CJ, Tsou JH, Bonci A, Ikemoto S, 2015. Mesopontine median raphe regulates hippocampal ripple oscillation and memory consolidation. Nat Neurosci 18, 728–735. DOI: 10.1038/nn.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Westenbroek C, Den Boer JA, Ter Horst GJ, 2003. Gender-specific effects of social housing on chronic stress-induced limbic Fos expression. Neuroscience 121, 189–199. DOI: 10.1016/s0306-4522(03)00367-1. [DOI] [PubMed] [Google Scholar]
  58. Yan Y, Newman AH, Xu M, 2014. Dopamine D1 and D3 receptors mediate reconsolidation of cocaine memories in mouse models of drug self-administration. Neuroscience 278, 154–164. DOI: 10.1016/j.neuroscience.2014.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yue JL, Yuan K, Bao YP, Meng SQ, Shi L, Fang Q, Guo XJ, Cao L, Sun YK, Lu TS, Zeng N, Yan W, Han Y, Sun J, Shi J, Kosten TR, Xue YX, Wu P, Lu L, 2022. The effect of a methadone-initiated memory reconsolidation updating procedure in opioid use disorder: A translational study. EBioMedicine 85, 104283. DOI: 10.1016/j.ebiom.2022.104283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zangenehpour S, Chaudhuri A, 2002. Differential induction and decay curves of c-fos and zif268 revealed through dual activity maps. Mol Brain Res 109, 221–225. DOI: 10.1016/s0169-328x(02)00556-9. [DOI] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

1
NIHMS1967081-supplement-1.docx (33.7KB, docx)

RESOURCES