Exploring ketamine’s reinforcement, cue-induced reinstatement, and nucleus accumbens cFos activation in male and female long evans rats

Abstract

Ketamine (KET), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, has rapid onset of antidepressant effects in Treatment-Resistant Depression patients and repeated infusions are required to sustain its antidepressant properties. However, KET is an addictive drug, and so more preclinical and clinical research is needed to assess the safety of recurring treatments in both sexes. Thus, the aim of this study was to investigate the reinforcing properties of various doses of KET (0-, 0.125-, 0.25-, 0.5 mg/kg/infusion) and assess KET’s cue-induced reinstatement and neuronal activation in both sexes of Long Evans rats. Neuronal activation was assessed using the protein expression of the immediate early gene cFos in the nucleus accumbens (Nac), an important brain area implicated in reward, reinforcement and reinstatement to most drug-related cues. Our findings show that KET has reinforcing effects in both male and female rats, albeit exclusively at the highest two doses (0.25 and 0.5 mg/kg/infusion). Furthermore, we noted sex differences, particularly at the highest dose of ketamine, with female rats displaying a higher rate of self-administration. Interestingly, all groups that self-administered KET reinstated to drug-cues. Following drug cue-induced reinstatement test in rats exposed to KET (0.25 mg/kg/infusion) or saline, there was higher cFos protein expression in KET-treated animals compared to saline controls, and higher cFos expression in the core compared to the shell subregions of the Nac. As for reinstatement, there were no notable sex differences reported for cFos expression in the Nac. These findings reveal some sex and dose dependent effects in KET’s reinforcing properties and that KET at all doses induced similar reinstatement in both sexes. This study also demonstrated that cues associated with ketamine induce comparable neuronal activation in the Nac of both male and female rats. This work warrants further research into the potential addictive properties of KET, especially when administered at lower doses which are now being used in the clinic for treating various psychopathologies.

1. Introduction

In the United States, Major Depressive Disorder (MDD) is the most common mood disorder. According to the relatively recent 2021 SAMHSA report (SAMHSA, 2021), approximately 21 million adults in the US—which corresponds to 8.3% of all US adults—had at least one major depressive episode, with much higher prevalence among women (10.3%) compared to men (6.2%). The economic impact of MDD continues to rise, and the total attributed cost just this past year exceeded $382.4 billion (Greenberg et al., 2023). For the past 50–60 years, MDD treatment has relied mostly on behavioral therapy and/or some form of pharmacological treatment, such as the selective serotonin reuptake inhibitors (SSRI) or serotonin norepinephrine reuptake inhibitors (SNRI). However, these pharmacological treatments are not always very effective in all patients and take a long time to improve mood.

Berman et al. (2000) were first to show that ketamine (KET), a noncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist, exhibits rapid antidepressant effects in treatment resistant depressed (TRD) patients. In this population, a single intravenous (i.v.), slow infusion of low-dose KET (0.5 mg/kg) administered over 40 min improved depressive symptoms within as little as 2 h (Berman et al., 2000). Subsequent studies replicated these findings (Zarate et al., 2006; Vidal et al., 2018; Bahji et al., 2021). However, most TRD patients require repeated infusions of KET to increase the likelihood of response to treatment and achieving remission (Zheng et al., 2018; Murrough et al., 2013). Since major depression is usually comorbid with other psychopathologies, other studies have suggested that KET can also be a potential therapy for Alcohol and Substance Use Disorder (AUD/SUD) and Post Traumatic Stress Disorder (PTSD) (Dakwar et al., 2019, 2020; Abdallah et al., 2019; Grabski et al., 2022). It is however important to note that KET is an illicit drug used recreationally and was shown to cause serious cognitive and mood-related deficits, in addition to other serious negative health-related outcomes (Morgan et al., 2004a, 2004b, 2004c, 2004d, 2009a, 2009b, 2010; Chang et al., 2016). In fact, little has been done to examine the abuse potential of repeated exposure to KET despite its widespread clinical applications for Treatment-Resistant Depression (TRD) and various other pathologies.

Another important variable not seriously considered in the past is whether sex differences exist in the reinforcing and potential addictive properties of ketamine. It is well established now that there are clear gender differences in mood and AUD/SUDs, where females differ in onset compared with males, progress more rapidly through the stages of addiction, and have more comorbid psychiatric disorders (Brady et al., 1999; Bobzean et al., 2014; Lai et al., 2015). Studies have also shown both activational and organizational sex differences in the mechanisms underlying the development of psychiatric disorders (Becker et al., 2016; Saland et al., 2017, 2018; McHenry et al., 2014). Related to KET, we have previously demonstrated that, when compared to males, female rodents are more sensitive to its antidepressant effects, and provided evidence that cycling ovarian hormones mediate, in part, this high sensitivity (Carrier et al., 2013; Dossat et al., 2018; Saland et al., 2016).

Earlier studies clearly demonstrated the importance of environmental context (home vs. novel environment) in the reinforcing properties of various doses of KET (De Luca et al., 2011; Venniro et al., 2015). These studies were however conducted only in male rats and thus expanding this to female subjects is warranted. Our previous work showed that a low dose of KET (0.1 mg/kg/Infusion) is reinforcing in male and female rats in proestrus, but not in females in diestrus, suggesting that cycling ovarian hormones may have mediated female sensitivity to the reinforcing properties of low doses of KET (Wright et al., 2017). In another study, when we assessed KET reinforcing properties at a higher dose (0.5 mg/kg/Infusion), female rats administered more KET than males. Further, within the context of stress, females—but not males—with a history of chronic stress-exposure displayed greater motivation to self-administer KET (Strong et al., 2019; Wright et al., 2019).

Since drugs of abuse hijack natural reward pathways, understanding how KET mediates neuroadaptations in the Nac—considered the hub for motivated behavior and reward—is of particular interest. The Nac receives modulatory dopaminergic input from the ventral tegmental area (VTA), as well as converging glutamatergic input from the amygdala, hippocampus, thalamus, and prefrontal cortex (PFC) (Scofield et al., 2016; Gardoni et al., 2015). Clinical fMRI studies and other preclinical studies provided evidence that some of KET’s antidepressant effects are mediated, at least in part, through recruitment of brain circuits including projections from the PFC to Nac (Abdallah et al., 2017, 2018; Chen et al., 2019). These projections have been shown play a very important role in the salience of drug-related cues (Scofield et al., 2016; Rogers et al., 2008). The Nac is subdivided into the core and shell subregions, which have different roles in motivated and goal-oriented behaviors for natural rewards and drugs of abuse based on their input and output targets (Scofield et al., 2016; Ambroggi et al., 2011). Regarding KET, little research has been done to examine involvement of the NAc core and shell during cue-induced reinstatement, an animal model of relapse. Thus, one of the aims in this work was to determine whether KET cues induce reinstatement and use cFos, a marker of neuronal activity, to assess whether the core and/or shell neurons get activated after the reinstatement to KET cues. Accordingly, this study thus sought to determine the reinforcing properties of various doses of KET, as well as neuronal activity in the NAc Core and Shell following cue-induced reinstatement in both sexes of Long Evans rats.

2. Experimental procedures

Experimental procedures were used in our previous publications with minor changes to align them with the aims of this study (Strong et al., 2017, 2019; Wright, 2017, 2019).

2.1. Animals and housing

Adult wild type male and female Long Evans rats (60 days old at the beginning of experiment) originated from our own breeding colony where wild type Long Evans rats (Charles River, Wilmington, MA) were crossed with either Drd1a-iCre (LE-Tg(Drd1a-iCre)3Ottc) or Drd2-iCre rats (LE-Tg(Drd2-iCre)1Ottc) (NIDA transgenic rat project; Rat Resource and Research Center [RRRC], Columbia, MO). Rats were maintained in a temperature- and humidity-controlled room under a 12-h light/dark cycle (lights on: 9 p.m.; Lights off: 9 a.m.). Rat pups were weaned on postnatal day 21, and genotyped. Wild type rats were pair-housed with same-sex littermates (43 × 21.5 × 25.5 cm Plexiglass cage) with environmental enrichment (one 4-inch PVC pipe). Food and water were provided ad libitum. All experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals, and all protocols were approved by the Florida State University Institutional Animal Care and Use Committee.

Adult male and female rats (initially ten to twelve weeks old, weighing 333–524 g and 211–336 g, respectively) were used. One week prior to the start of experimental procedures, rats were transferred to housing suites operated under a reverse light/dark cycle (lights on: 7 p.m.; lights off: 7 a.m.) which was maintained throughout the course of the study. Animals were weighed and handled daily to acclimate to the experimenter. Each experimental group was represented by at least five different breeder pairs from the colony and behavioral experiments were conducted in 9 cohorts. A total of 89 rats were used in the current study; 1 male and 1 female rat were removed from the study after having to be terminated for unknown illnesses that caused them to lose >20% of their body weight; 4 male rats were removed from the study for having catheter patency issues. 1 male and 5 female rats were removed after an experimenter error in drug delivery.

2.2. Drugs

For surgery, isoflurane (Isothesia, Henry Schein: 029405) was used for anesthesia with the EZ-AF9000 Auto Flow System (E-Z Systems, EZ-AF9000). Betadine surgical scrub (Covetrus North American: 038248) was used as an antiseptic microbicide, followed by 70% ethanol (Koptec [Decon Labs]: V1001GTP) for sterilization of surfaces. Heparin (HEP, 50 units/mL; Alfa Aesar [VWR International]: AAA16198-03) and ampicillin (AMP, 30 mg/mL; USP, VWR Life Sciences [VWR International]: 97061-442) were both dissolved in 0.9% sterile saline (SAL) solution to fill the catheter tubing during surgery, administered intravenously to maintain patency, and prevent post-operative infection. Bupivacaine (2.5 mg/mL; Sigma-Aldrich, B5274) was administered post-operatively as a topical analgesic. Ketoprofen (5 mg/mL; Ketofen, Zoetis [Patterson Veterinary]: 07-803-7389) was injected at a volume of 1 mL/kg subcutaneously for post-operative analgesia.

For Self-Administration (SA) experiments, rats were flushed only with HEP in SAL (50 U/mL) to maintain patency. KET hydrochloride (Ketasthesia^®, racemic, Covetrus North American: 071069) was diluted from a 100 mg/ml stock solution in 0.9% sterile SAL (Intermountain Life Sciences [VWR International]: 75832-226) and administered intravenously at doses of 0.125-, 0.25-, or 0.5 mg/kg/infusion in a 50 μL volume. These doses were similar to those previously reported in male rats (De Luca et al., 2011; Dietz et al., 2008). Xylazine hydrochloride (2.5 mg/mL in SAL; ThermoScientific [VWR International]: AAJ62394-06) was administered intravenously at a volume of either 0.05 (females) or 0.1 (males) mL to assess catheter patency.

For transcardial perfusion, sodium pentobarbital (Soccumb, Covetrus North American: 017285) was diluted 1:1.5 in 0.9 % sterile SAL and administered via a single intraperitoneal (i.p.) injection in a volume of 1 mL/kg to deeply anesthetize rats.

2.3. Locomotor response in a novel environment

Before surgery, rats underwent an initial 1-hr novelty-induced locomotor activity test as previously described (Dietz et al., 2008; Kabbaj, 2006). This test allows categorization of rats into high- or low-responders to novelty based on locomotor activity scores above or below the median score for each cohort. This test was used when assigning and balancing experimental groups to ensure rats with differential responses to novelty were equally distributed between groups, as individual differences in response to novelty, in general, can influence responsivity to drugs of abuse.

2.4. Jugular catheterization

Was performed as previously described by our group (Wright et al., 2017, 2019; Strong et al., 2019). Briefly, rats were induced (5%) and maintained (3–4%) with isoflurane on pure oxygen (2 L/min); Rats were flipped over onto their back and the location of the right external jugular vein was identified visually by heartbeat. Starting at the site of the heartbeat, a straight 2 cm incision was made anterior toward the head, anda subcutaneous vascular access button with magnetic backport (Intech Laboratories, Plymouth Meeting, PA) was connected to a 1 mL syringe pre-filled with HEP/AMP, then an i.v. catheter (Intech Laboratories) was attached to the subcutaneous fastener on the backport. The catheter was inserted into the jugular vein via a small incision and secured to the vein. Bupivacaine was applied to the suture site, Ketoprofen was injected, and rats were given 5–7 days of once, daily post-operative care with 0.2 mL HEP/AMP before starting self-administration (SA) sessions. Rats were flushed daily with 0.1 mL HEP before and after SA sessions to maintain patency. Following sessions 10 and 20, patency was tested with xylazine, followed by 0.2 mL HEP to clear the catheter. Rapid loss of muscle tone was an indicator of patency; rats that maintained tone were no longer considered patent. Rats were removed from SA, a new catheter was installed on the left external jugular vein and resumed SA following another recovery period of at least 3 days. 4 rats were removed from the experiment because they failed a second patency test.

2.5. Ketamine self-administration acquisition, extinction, & reinstatement

After recovery from catheter surgeries, male and female rats were run in operant chambers (30.5 × 24.1 × 21.0 cm; Med Associates) housed inside sound attenuating boxes at least 1-hr after the onset of the dark-cycle in a room illuminated with red light. The left chamber wall was equipped with two nose-poke holes on the left and right with a cue-light inside, with a house light centered at the top of the opposing wall. On the box exterior, a syringe pump (3.33 rpm single-speed; Med Associates) loaded with a pre-filled 10 mL syringe containing ketamine or saline solution connected to a single-channel 22-gauge plastic swivel mounted on a magnetic tether arm with counterweight via 22-gauge tubing (Instech Labs) fixed to the chamber’s plexiglass ceiling. The tether arm connected the delivery line to the rat’s catheter-backport assembly allowing for full range of motion within the chamber while receiving Infusions. Rats remained tethered for all experimental sessions.

At the start of each SA session, illumination of the house-light signaled availability of KET or SAL. Rats self-administered one of three doses of KET (0.125, 0.25, or 0.5 mg/kg/Infusion) or SAL during a 2-hr session. A nose-poke to the active hole resulted in a single 50 μL i.v. infusion of KET/SAL, during which time the intra-operandum cue-light turned on for the duration of the infusion. The house light also turned off following a correct response to the active nose-poke hole, indicating that KET/SAL was now unavailable following the single infusion, and remained off for the remainder of a 20-sec timeout period. Nose-pokes made to the inactive side resulted in no programmed consequence and discrimination between active and inactive was used as a measure of learning. All rats self-administered under fixed ratio (FR) 1, 3, and 5 schedules of reinforcement across 20 2-h SA sessions. Following FR1, FR3 and FR5 sessions, rats began 10 2-h daily sessions of extinction (EXT) training, where the syringe pump and house light were turned off, and both active and inactive nose-pokes had no programmed consequences. Finally, rats underwent a single 2-h cue-induced reinstatement test, where drug-paired cues (house light, cue light, pump) were returned to the operant chamber, but no KET/SAL solution was available. All responses to both nose-poke holes were recorded during all experimental sessions using Med Associates software.

2.6. Tissue collection & immunohistochemistry

Rats were terminated at the end of the 2-h reinstatement test. We used immunohistochemistry (IHC) to assess NAc cFos protein expression. Rats received an i.p. injection of sodium pentobarbital to deeply anesthetize them, then were transcardially perfused with ice-cold 0.2 M phosphate buffered SAL (PBS) followed by ice-cold 4% paraformaldehyde (PFA) in 0.2 M PBS. Brains were extracted, post-fixed in 4% PFA in 0.2 M PBS at 4 °C for 24-hrs, and transferred to 0.01% sodium azide in 0.2 M PBS at 4 °C. Serial 40 μm NAc coronal slices were collected from the SAL and KET (0.25 mg/kg/infusion) groups with a Leica vibratome (VT1200S) and stored in 0.01% sodium azide in 0.2 M PBS at 4 °C.

Tissue is washed 3 times with 0.2 M PBS (5 min), incubated in 0.3% Triton X-100 in 0.2 M PBS (1 h), then placed in blocking buffer containing 5% normal goat serum (NGS), 5% bovine serum solution (BSA), and 0.3% Triton X-100 in 0.2 M PBS (1-hr). Tissue was placed in blocking buffer containing primary antibody (anti-cFos, rabbit, monoclonal, 1:500, Cell Signaling #2250) at 4 °C for 48-hrs. Tissue was removed from primary antibody, washed three times in 0.3% Triton X-100 in 0.2 M PBS (5 min), and placed in blocking buffer containing secondary antibody (Alexa Fluor Plus 594, donkey anti-rabbit, 1:500, Invitrogen #A21424) in 0.2 M PBS on a rocker at 4 °C overnight. Tissue was washed twice with 0.2 M PBS (5 min), mounted on Superfrost Plus Microscope Slides (Fisher: #12-550-15), and labeled with DAPI (NucBlue Fixed Cell ReadyProbes Reagent [ThermoFisher: R37606]) for 30 s. Tissue was covered with ProLong^™ Gold Antifade Mountant medium (ThermoFisher: P36930) and slides were cover-slipped with micro cover glass (VWR 48393; No. 1.5 - 24 × 50 mm).

2.7. Image acquisition & quantification

Image acquisition for quantification was performed on a Zeiss LSM780 laser-scanning confocal microscope at 20× objective magnification. Sixteen-bit snapshots were collected during excitation with the 405-laser (DAPI) and the 594-laser (cFos). Quantification of cFos was done with images taken throughout the NAc core and shell. Location was confirmed with physical topography of the section matched against the rat brain atlas, then subregions were determined by their relative position to the anterior commissure. Four tissue sections were used per animal, with three to four images were taken per hemisphere of each section. A minimum of 12 images per subregion per rat were used for counting. Images were loaded into ImageJ (NIH Image, 1.8.0_172), and within each image total cFos-positive nuclei were manually quantified by two scorers blind to the experimental conditions.

2.8. Statistical analyses

Behavioral data was analyzed with either GraphPad Prism (version 8.11) or RStudio (version 3.5.1), and IHC quantification was analyzed using RStudio. Analysis of variance (ANOVA) was performed in RStudio using a linear mixed-model (LMM) framework with the lmerTest and emmeans packages. Group factors during SA acquisition, EXT, and reinstatement consisted of Sex (male, female), Dose (0.0, 0.125, 0.25, 0.5), Response (active, inactive), and Session. Sex and Dose were between-subjects factor, and Response and Session were within-subjects factor. For LMM ANOVAs, time was treated as a linear vector to analyze the effect of Session using a repeated measures mixed-model framework. FR SA and EXT were analyzed separately due to the difference in response requirement for drug availability/reinforcement. For Infusions, Three-way LMM ANOVA was used (Sex, Dose, Session). For analysis of nose-poke responses, Four-way LMM ANOVA was used (Sex, Dose, Response, Session). Where a main effect of Dose was present during SA, each KET dose was analyzed separately. For Reinstatement, analysis only considered EXT day 10 and the reinstatement test. For cFos, Three-way mixed model ANOVA was used (Sex, Dose, Region [core, shell]). To determine whether an increase/decrease in intake or responses occurred (SA, EXT, Reinstatement), the parameter estimates of the LMM output were observed. Statistical significance was set at α = 0.05 and outputs were considered significant when p < 0.05. Statistically significant main effects or interactions were followed up with multiple comparison post-hoc tests with appropriate corrections. A Pearson correlation was run for active responses and cFos expression in the core and shell NAC during reinstatement. All data is represented as mean ± SEM. For comparisons, a total of N = 77 (37 male, 40 female rats) rats were used with n = 8–12/group. Grubb’s outlier test was conducted for each group session and within-subject comparison; data that were statistical outliers for both were removed.

3. Results

3.1. Acquisition of KET self-administration – ketamine elicits differential responses based on sex and dose

3.1.1. FR1 schedule

For number of infusions (Fig. 1A-D), main effects of Dose (F_3,71 = 20.94, p < 0.0001) and Session (F_9,629 = 30.85, p < 0.0001) were observed, along with interactions of Sex x Dose (F_3,71 = 2.747, p < 0.0001) and Dose x Session (F_27,629 = 7.165, p < 0.0001). Post-hoc analyses revealed that the number of infusions received significantly increased across FR1 sessions in all KET—but not SAL—administering groups, regardless of sex (p < 0.05, session 10 vs 1). Area under the curve (AUC) was determined for FR1 sessions 1–10 and compared between dose groups within male and female cohorts as a cumulative measurement of drug response throughout the acquisition period (Fig. 2A). Here, infusion AUC values were greater for 0.25 and 0.50 mg/kg/inf KET-administering male and female rats relative to SAL groups (all ps < 0.05 vs. SAL) and otherwise differed between KET groups in a dose- and sex-dependent manner (p < 0.0001 KET vs. KET; Sex: F_(1,708) = 0.6721, p = 0.4126; Dose: F_(3,708) = 25.24 p < 0.0001; Interaction: F_(3,708) = 3.383, p = 0.0179). Specifically, male rats self-administering KET at 0.25 and 0.5 mg/kg/inf took significantly more infusions compared to male rats self-administering KET at 0.125 mg/kg (all ps < 0.01), whereas only female rats self-administering 0.5 mg/kg/inf KET exhibited significantly increased infusions relative to those administering 0.125 mg/kg/inf (p < 0.001). No differences were observed between the highest doses administered for either males or females (p > 0.05). Between-sex comparisons found that only at the highest dose (0.5 mg/kg/inf) did females and males differ in number of infusions received, with females trending toward more infusions than males across the FR1 period (p = 0.0585).

Fig. 1.

Mean number (±SEM) of infusions for independent groups of Long Evans male and female rats self-administering saline (A) and ketamine (0.125 (B), 0.25 (C), 0.5 (D) mg/kg) under various fixed ratios (FR) FR1, FR3 and FR5. The FR was progressively increased from 1 (sessions 1–10) to 3 (sessions 11–15) to 5 (sessions 16–20). (E).

Fig. 2.

AUC values for infusions received across FR1 (A), FR3 (B), and FR5 (C) sessions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. saline or ketamine.

For responses (Fig. 3A-D), main effects of Sex (F_1,71 = 4.260, p = 0.0427), Dose (F_3,71 = 24.84, p < 0.0001), Response (F_1,1328 = 635.8, p < 0.0001), and Session (F_9,1345 = 1328, p < 0.0001) were observed. Interactions of Sex x Dose (F_3,71 = 3.872, p = 0.01268), Dose x Response (F_3,1328 = 93.50, p < 0.0001), Dose x Session (F_27,1328 = 5.552, p < 0.0001), Response x Session (F_9,1328 = 7.883, p < 0.0001), Sex x Dose x Response (F_3,1328 = 7.755, p < 0.0001), Sex x Dose x Session (F_27,1328 = 2.309, p < 0.0001), and Dose x Response x Session (F_27,1328 = 2.1258, p = 0.0007) were observed. Comparison of AUC values across the FR1 period for male and female groups revealed a significant effect of Dose, but not Sex, in active responding (Fig. 4A; Dose: F_(3,700) = 22.84, p < 0.0001; Sex: F_(1,700) = 3.071, p = 0.801; Interaction: F_(3,700) = 2.615, p = 0.0502). In both males and females, rats self-administering 0.25 and 0.50 mg/kg/inf—but not the lowest dose (p > 0.05)—responded significantly more than rats receiving SAL (p < 0.05). Females at the highest 2 doses also responded more than those administering 0.125 mg/kg/inf (p < 0.001), with no apparent difference in active responding between these doses (p > 0.05, 0.25 vs. 0.50 mg/kg/inf). Conversely, 0.25 mg/kg/inf KET males responded significantly more than those receiving 0.125 (p < 0.0001) or 0.50 mg/kg/inf (p = 0.0130) KET. Between-sex, only females self-administering 0.5 mg/kg/inf KET made significantly more active responses than their male counterparts (p = 0.0046). Males in 0.25 (p = 0.0139, session 10 vs 1) and 0.50 mg/kg/inf (p = 0.0144, session 8 vs 1) groups significantly increased active responding across sessions—a trend only observed in females administering 0.50 mg/kg/inf KET (p = 0.0093, session 10 vs 1). Interestingly, females receiving 0.50 mg/kg/inf exhibited significant increases in inactive responses relative to SAL females throughout acquisition (p = 0.0129). These findings were confirmed in AUC analyses for inactive responses which found main effects of Dose, Sex, and a Dose × Sex interaction (Fig. 4D; Dose: F_(3,701) = 8.371, p < 0.0001; Sex: F_(1,701) = 4.226, p = 0.0402; Interaction: F_(3,701) = 2.859, p = 0.0362). Females receiving 0.50 mg/kg/inf made significantly greater inactive responses than their male counterparts (p = 0.0012), as well as females receiving either SAL or 0.125 mg/kg/in (ps < 0.005).

Fig. 3.

Mean number (±SEM) of active and inactive nose pokes for independent groups of Long Evans male and female rats self-administering saline (A) and ketamine (0.125 (B), 0.25 (C), 0.5 (D) mg/kg) under various fixed ratios (FR) FR1, FR3 and FR5. The FR was progressively increased from 1 (sessions 1–10) to 3 (sessions 11–15) to 5 (sessions 16–20).

Fig. 4.

AUC values for active and inactive responses made across FR1 (A,D), FR3 (B, E), and FR5 (C, F) sessions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. saline or ketamine.

3.1.2. FR3

For Infusions (Fig. 1A-D), a main effect of Dose (F_3,71 = 17.01, p < 0.0001) and a Sex × Dose interaction (F_3,71 = 3.867, p = 0.0127) were observed. AUC analyses further identified dose-dependent increases in infusions in male and female rats, where only 0.25 mg/kg/inf males and 0.50 mg/kg/inf females received significantly more KET when compared to their 0.125 mg/kg/inf (Fig. 2B; Males: p = 0.0095; Females: p = 0.0184) and SAL (Males: p = 0.0075; Females: p = 0.0052) same-sex counterparts. No sex differences in AUC values were observed at any dose examined in either sex (p > 0.05 Females vs. Males; AUC 2-Way ANOVA: Dose: F_(3,313) = 8.757, p < 0.0001; Sex: F_(1,313) = 0.06693, p = 0.7960; Interaction: F_(3,313) = 1.854, p = 0.1374).

For responses (Fig. 3A-D), main effects of Dose (F_3,71 = 13.96, p < 0.0001) and Response (F_1,635 = 240.6, p < 0.0001) were observed. Interactions of Sex x Dose (F_3,71 = 3.662, p = 0.0163), Dose x Response (F_3,635 = 45.76, p < 0.0001), and Sex x Dose x Response (F_3,635 = 8.065, p < 0.0001) were observed as well. As with infusions, dose-dependent increases in FR3 AUC values for active responses were identified in a sex-specific manner (Fig. 4B; Dose: F_(3,313) = 7.455, p < 0.0001; Sex: F_(1,313) = 0.005079 p = 0.9432; Interaction: F_(3,313) = 1.697, p = 0.1675). Here, compared to same-sex controls receiving SAL or 0.125 mg/kg/inf KET, active responding was significantly greater only in males self-administering 0.25 mg/kg/inf (p = 0.0212 vs. SAL, p = 0.0249 vs. 0.125 mg/kg/inf) and females receiving 0.50 mg/kg/inf (p = 0.0059 vs. SAL, p = 0.0208 vs. 0.125 mg/kg/inf). No differences in active responding between males and females were observed under a FR3 schedule of reinforcement. No differences in inactive responding were noted between dose groups in either males or females. Female rats in all KET groups—but not SAL (p = 0.2267)—had significantly higher active compared to inactive responses (all ps < 0.01). In male rats, only 0.25 and 0.5 mg/kg/inf KET groups exhibited significantly higher active compared to inactive responses (p < 0.0001). A main effect of Dose was observed for inactive response AUC values during FR3 (Fig. 4E; Dose: F_(3,354) = 3.387, p = 0.0183; Sex: F_(1,354) = 1.986, p = 0.1596; Interaction: F_(3,354) = 2.116, p = 0.0979). As with FR1, only females receiving 0.50 mg/kg/inf responded more at the inactive nose poke compared to 0.50 mg/kg/inf-receiving males (p = 0.0147), and females receiving SAL (p = 0.0063), 0.125 mg/kg/inf (p = 0.0132), or 0.25 mg/kg/inf (p = 0.0203).

3.1.3. FR5

For infusions (Fig. 1A-D), a main effect of Dose (F_3,71 = 11.36, p < 0.0001) and a Dose × Session interaction (F_12,276 = 1.438, p = 0.0389) were observed. Further, a trend was observed for an effect of sex following examination of AUC values across FR5 sessions (Fig. 2C; Sex: F_(1,297) = 3.387, p = 0.0667; Dose: F_(3,297) = 286.7, p < 0.0001; Interaction: F_(3,297) = 18.21, p < 0.0001). Neither males nor females receiving 0.125 mg/kg/inf received significantly more KET infusions when compared to SAL rats (ps > 0.05, 0.125 vs. SAL). Both males and females exhibited within-sex step-wise increases in infusions received with each increasing dose of KET (all ps ≤ 0.001, KET vs. KET). With the increased effort requirement to receive drug, females received significantly more infusions than males at 0.25 (p < 0.0001) and 0.50 mg/kg/inf (p < 0.0001).

For responses (Fig. 3A-D), main effects of Dose (F_3,71 = 12.26, p < 0.0001) and Response (F_1,622 = 230.2, p < 0.0001) were observed. Interactions of Dose x Response (F_3,622 = 51.11, p < 0.0001) and Sex x Dose x Response (F_3,622 = 5.016, p < 0.0001) were observed. While post-hoc comparisons revealed that both 0.5 mg/kg/inf KET males and females made significantly more active responses when compared to their same-sex SAL controls when considered on a session-by-session basis (p < 0.05, sessions 16–20), AUC analyses only identified significantly greater active responding in females self-administering 0.5 mg/kg/inf under an FR5 schedule of reinforcement relative to both 0.125 mg/kg/inf (p = 0.0021) and SAL (p = 0.0102) female rats (Fig. 2C; Sex: F_(1,307) = 0.3678, p = 0.5447; Dose: F_(3,307) = 7.247, p = 0.0001; Interaction: F_(3,297) = 0.8699, p = 0.4570). No other differences were identified between male and female rats across doses. Main effects of Dose, Sex, and a Dose × Sex interaction were observed for inactive response AUC values across FR5 sessions (Fig. 4F; Dose: F_(3,346) = 7.298, p < 0.0001; Sex: F_(1,346) = 4.285, p = 0.0392; Interaction: F_(3,346) = 3.103, p = 0.0267). Here again, females receiving 0.50 mg/kg/inf responded more at the inactive nose poke compared to SAL- (p < 0.0001), 0.125 mg/kg/inf- (p < 0.0001), or 0.25 mg/kg/inf- (p = 0.0003) administering females, as well as males receiving 0.50 mg/kg/inf KET (p = 0.0009).

3.2. Extinction & reinstatement

Extinction data are shown in Fig. 5A-D. Responding was successfully extinguished in all male and female groups by the final extinction session (defined as the lack of within-sex statistical difference (>0.05) p relative to SAL control groups).

Fig. 5.

Mean number (±SEM) of active and inactive nose pokes for independent groups of Long Evans male and female rats during extinction from saline (A) and ketamine (0.125 (B), 0.25 (C), 0.5 (D) mg/kg) and their associated cues. Session 0 corresponds to the last day of acquisition, and sessions 1–10 are the extinction days.

Reinstatement data are presented in Fig. 5. There were main effects of Dose (F_3,69 = 3.778, p = 0.0143), Response (F_1,207 = 180.7, p < 0.0001), and Session (F_1,207 = 92.23, p < 0.0001). Interactions of Dose x Response (F_3,207 = 6.146, p = 0.0005), Dose x Session (F_3,207 = 8.535, p < 0.0001), Response x Session (F_1,207 = 77.46, p < 0.0001), and Dose x Response x Session (F_3,207 = 4.740, p = 0.0032) were observed as well. Post-hoc analyses found that KET and SAL groups made significantly more active than inactive responses during the reinstatement test (all doses, p < 0.001). However, regardless of sex only rats that received KET made significantly more active responses during the reinstatement test compared to EXT day 10 (all p < 0.001)—SAL rats did not (p > 0.05). Moreover, all rats receiving KET made significantly more active responses during the test session compared to SAL (all p < 0.0001, KET vs. SAL), suggesting that only rats self-administering KET reinstated responding to cues (ps < 0.05). No other group differences were detected during reinstatement. Inactive responding during either the final day of extinction or reinstatement session did not differ between male and female rats at any dose (Fig. 6B; Sex: F_(1,69) = 0.191, p = 0.664; Dose: F_(3,69) = 0.734, p = 0.535; Session: F_(1,69) = 3.573, p = 0.063; Sex x Dose: F_(3,69) = 0.211,p = 0.889; Sex x Session: F_(1,69) = 1.681, p = 0.199; Sex x Dose x Session: F_(3,69) = 1.888, p = 0.140). However a Dose × Session interaction was observed (F_(3,69) = 3.189, p = 0.029), such that animals self-administering 0.25 mg/kg/inf responded more at the inactive aperture during reinstatement than during the final extinction session (p < 0.05), regardless of sex.

Fig. 6.

Mean number (±SEM) of active (A) and inactive (B) nose pokes for independent groups of Long Evans male and female rats during reinstatement of cues previously associated with saline and ketamine (0.125, 0.25, 0.5 mg/kg) self-administration. EXT D10 = active responses during the last day of extinction in the absence of saline or ketamine and their associated cues; Reinstate = active responses during the one session of reinstatement in the presence of cues. *p < 0.05 Reinstate vs. EXT D10.

3.3. cFos expression in the nucleus accumbens

Data are shown in Fig. 7. Fig. 7A depicts representative images of the immunohistochemistry. Overall, there were main effects of Dose (F_1,19 = 6.105, p = 0.0231) and Region (F_1,490 = 154.0, p < 0.0001) (Fig. 7B). A Dose × Region Interaction (F_1,490 = 15.03, p = 0.0001) was observed. Post-hoc analysis showed that rats had significantly more cFos expression in the core compared to shell (both, p < 0.0001). As well, significantly more cFos expression was observed in the Nac core of rats that reinstated to KET cues compared to SAL controls (p = 0.0023). There were no differences between cFos expression in the shell between KET and SAL groups (p = 0.1385). Pearson analysis revealed no significant correlations between active responses during reinstatement and cFos expression in the shell (r² = 0.077; p = 0.408) or in the core (r² = 0.002; p = 0.891) subregions of Nac.

Fig. 7.

cFos protein expression in the nucleus accumbens core and shell subregions. (A) Comparative images of cFos protein expression in the NAc following cue-induced reinstatement to saline (SAL) and ketamine (KET, 0.25 mg/kg) (B) Quantification of cFos protein expression in the core and shell subregions of the NAc following cue-induced reinstatement to saline and ketamine (0.25 mg/kg). **p < 0.01 vs. SAL, ****p < 0.0001 Core vs. Shell.

4. Discussion

The purpose of this study was to assess the reinforcing properties and cue-induced reinstatement of various doses of KET in male and female Long Evans rats. We also examined cFos protein expression in the Nac core and shell as a marker of neuronal activity following reinstatement. We reported some sex and dose-dependent differences in the reinforcing properties of KET, but no differences during extinction to KET cues. No sex differences in reinstatement were observed at any KET dose. Further, KET-associated cues increased cFos expression in the core—but not the shell—of the Nac in both sexes. Within the Nac, there was more cFos protein expression in the core than in the shell.

During the varied fixed ratio schedules, we observed sex- and dose-dependent differences in the reinforcing properties of 3 doses of KET. Our results suggest that under FR1 schedules of reinforcement, KET is reinforcing in both male and female rats when self-administered only at the highest two doses (0.25 and 0.5 mg/kg/infusion) examined. Interestingly, with increasing effort requirements, sex differences in dose response became more apparent. Specifically, males increased active responding under an FR3 schedule of reinforcement selectively for 0.25 mg/kg/inf KET, but not the highest dose—this increased responding was not maintained when effort requirements were increased to FR5. Conversely, active responding was significantly increased and maintained in females self-administering 0.5 mg/kg/inf KET (but not lower doses) across both FR3 and FR5 schedules of reinforcement. While no sex differences in self-administration of ketamine were observed when effort requirements were low, in agreement with our previous findings in Sprague-Dawley rats (Wright et al., 2017), the present work demonstrates a dose-dependent sex difference in the amount of effort rats will expend to receive equivalent doses of KET. One notable limitation of our study is the absence of consideration for the influence of the estrous cycle on ketamine self-administration. Indeed, in Sprague-Dawley rats, we did observe an impact of the estrous cycle at a ketamine dose of 0.1 mg/kg/infusion, where proestrus female rats self-administered more ketamine than diestrus rats (Wright et al., 2017). It’s worth noting, however, that in that protocol, rats underwent self-administration sessions intermittently, aligned with specific estrous cycles, and were limited to 50 infusions per session. In our current investigation with Long-Evans rats, where daily ketamine exposure occurred without a cap on the number of infusions, the variability observed in female rats was comparable to that of males and therefore, we believe, estrous cycle may not have affected our present data. These findings contrast with the increased female sensitivity to KET reported for KET rewarding properties in rats as assessed in the conditioned place preference test (Strong et al., 2017; Schoepfer et al., 2019) and for sensitization to its locomotor activating effects (Schoepfer et al., 2019). Importantly, this sex difference has also been reported for KET antidepressant and anti-anhedonic effects (Carrier et al., 2013; Dossat et al., 2018; Saland et al., 2016) which suggest some differences between KET reinforcing and antidepressant properties.

In this work, when rats were self-administering KET under an FR1 ratio, we elucidated different sensitivities in male and female rats to the reinforcing properties of KET. This was most notable at the highest dose of KET (0.5 mg/kg/inf) which elicited higher infusions and active responses in female rats compared to male rats, which was is in line with our previous work (Strong et al., 2019); however, males responded significantly more to the KET dose of 0.25 mg/kg/inf relative to all other doses examined within-sex, a finding not previously reported. In De Luca and Badiani’s investigation of the effects of context on KET self-administration in Sprague-Dawley rats (De Luca et al., 2011), they reported that KET at 0.5 mg/kg/inf produced higher number of responses and infusions compared to 0.25- and 0.125 mg/kg/inf in both resident and non-resident conditions. Our findings in males deviate slightly from theirs, likely due to numerous experimental disparities. For instance, while the experimenters in their research facilitated acquisition by priming rats, we did not. Additionally, they implemented a cap on the number of infusions, whereas no such cap was employed in our investigation. Furthermore, the rats in their study were subjected to a distinct reinforcement schedule, a saline control was absent, and they utilized a different rat strain. There were no significant differences between active responses and infusions for the 0.125 mg/kg KET and SAL in either sex, which suggest that this dose is not reinforcing in rats. Taken together, these results then suggest that the 0.25- and 0.5 mg/kg/inf KET doses were reinforcing to both males and females, but that the highest dose was significantly more reinforcing in females than in males. It is worth noting that, in Sprague-Dawley rats, under an intermittent paradigm of KET intravenous SA (SA every fourth day), we have previously shown that KET had reinforcing properties in both male and female rats in proestrus (Wright et al., 2017). This suggests, as shown for other drugs such as cocaine and opioids, that intermittent access to KET may have greater effects as compared to continuous KET access (Algallal et al., 2020; Bender et al., 2023; Calipari et al., 2013; Miczek et al., 2011; Nicolas et al., 2019, 2021).

When the work requirement was increased under FR3 and FR5 schedules of reinforcement, our results showed clear sex differences. The 0.5 mg/kg/inf KET dose elicited the most active responses and infusions in females compared to males—most notably during FR5. As shown throughout FR1, the 0.25 mg/kg/inf KET dose elicited the most infusions under FR3 -but not FR5-and active responses in males compared to females. This continued upward trend in active responses suggests that female rats may have a drug-vulnerable phenotype that is potentially predisposing them to self-administer more KET (Piazza et al., 2000). It is however worth mentioning that female rats self-administering the highest dose of KET (0.5 mg/kg/inf) were the only group to have significantly greater inactive responding as compared to any other group. Inactive responses increased across FR1 sessions and were maintained during FR3 and FR5. While it is possible that repeated intake of large doses of KET in each session could have led to cognitive deficits, it is also possible that this increase in non-specific responding could be the result of an overall increase in locomotor activity and stereotypic behaviors, similar to what is observed with other drugs such as methamphetamine (Hsieh et al., 2014). As shown for methamphetamine, an influx of KET may have resulted in dopaminergic dysfunction, and persistent stimulus-independent dopamine release resulting in aberrant attribution of salience to the inactive nose-poke hole (Howes et al., 2009). It would be of interest in future studies to determine whether these nonspecific responses in females result from cognitive impairment or some form of stimulant-induced psychosis.

During the 2-h reinstatement test, dose was the most important factor. Rats that self-administered KET made significantly more active responses during the reinstatement test compared to the last day of EXT, while rats self-administering SAL rats did not, suggesting that rats with a history of KET—regardless of dose—reinstated to its cues. Additionally, all KET rats made significantly more active responses during the reinstatement test compared to rats self-administering SAL, suggesting that the KET cues were eliciting true drug-seeking behavior above basal levels of responding. In this study, no sex differences in reinstatement to KET cues were detected, suggesting that the reported sex differences in reinstatement to drugs of abuse such as cocaine to KET (Feltenstein et al., 2007, 2011; Fuchs et al., 2008) cannot be generalized.

To examine neuronal activation following reinstatement to cues, brains from rats in the SAL and 0.25 mg/kg/inf KET dose were selected and assessed for cFos expression levels in the Nac. At this dose, there were no sex differences in number of infusions and active responses AUC data for FR1, 3, or 5. As well, this dose was significantly different from saline in both sexes at FR1 (vs. 0.125 which was not), suggesting that it was reinforcing, and relatively equally so in males and females. We also did not use the 0.5 mg/kg/infusion as there were clear sex differences during acquisition and high inactive lever pressing in female rats which could have been linked to some effects of ketamine on cognitive function at this high dose. As such, we were interested in elucidating how cues activate the Nac subregions in both sexes following reinstatement to cues. Various input and output structures to the Nac core and shell differentially contribute to goal-directed behaviors. Interestingly, the core and shell have different functions in mediating incentive-cue responding. Core inactivation decreases responses to reward-associated cues, whereas shell inactivation increases responses to a non-reward-associated cues (Scofield et al., 2016; Ambroggi et al., 2011). Our results showed that KET-treated rats had significantly more cFos protein expression in both the core and shell as compared to the SAL group. KET cues, however, elicited differential cFos expression between the NAc core and shell, where cFos expression in the core was significantly higher in the KET group compared to the SAL group. Interestingly, there were no correlations between responding during reinstatement to KET and cFos expression in either region of the NAC. This could be explained by the fact that our dependent variable was whether cFos positive nuclei are present or not, and not the intensity of the signal in each neuron which may have shown some correlations with the reinstatement. In future studies, in situ hybridization is a method that permits assessment of cFos mRNA expression, which would be more likely to better correlate with response levels during reinstatement. Taken together, these results suggest that the core subregion is likely part of a circuitry that mediates reinstatement to KET cues. As a way of comparison with other drugs of abuse, following exposure to stimulants, such as cocaine, cFos expression was also highest in the Nac core (Neisewander et al., 2000; Wang et al., 2014), and cocaine-treated rats displayed significantly more Fos-positive nuclei in the core than their saline-yoked counterparts (Bastle et al., 2012).

Overall, our findings demonstrate that even low doses of KET may have addictive potential and that there are important sex differences in KET reinforcing properties at higher work requirements in females. Furthermore, KET seem to recruit similar brain structures recruited by other drugs of abuse such as cocaine.

Data availability

Data will be made available on request.