Depression carries the highest global burden of disease of any mental illness.1,2
Treatment options are limited due, in part, to its heterogeneous symptomatology and high comorbidity with other conditions including substance use disorders.3
The etiology of depression and substance use disorders is poorly understood, especially with respect to females who, despite having a twofold increased risk for depression, have been historically underrepresented in neuroscience research.4,5
Additionally, both human and rodent studies indicate that females escalate drug use more rapidly than males and may be more prone to relapse, depending on drug type.6,7
Preclinical research must address these intersecting factors underlying disease susceptibility.
The discovery of the rapid antidepressant effects of subanesthetic intravenous (i.v.) ketamine (KET) invigorated a field limited by monoaminergic antidepressant drugs that require several weeks of treatment to produce effects.8–10
However, KET’s therapeutic benefits are hindered by its abuse potential at high doses in humans,11
and the fact that rats readily self-administer KET.12–15
Questions regarding KET’s safety require careful consideration before widespread integration into medical practice as an antidepressant.9
This current work attempts to address two factors that may underlie susceptibility to KET’s effects: sex and prior exposure to chronic stress as a precipitating factor for both drug relapse and depression. We utilized the unpredictable chronic mild stress (CMS) model16,17
to induce a depressive-like behavioral profile in male and female rats, followed by i.v. slow, passive infusions of KET and subsequent operant KET self-administration to test the hypothesis that subjects—especially females—with a history of CMS and KET treatment would have an increased propensity to abuse KET.
Most behavioral models studying KET’s antidepressant-like effects have utilized intraperitoneal (i.p.) injections. In clinics, patients typically receive 0.5 mg/kg KET i.v. delivered over a 40-min period, two to three times per week.9
Therefore, to more closely recapitulate clinical approaches, we used an i.v. dose of 1.47 mg/kg/40 min, previously shown to match N-methyl-D-aspartate receptor occupancy in rats equivalently to clinically relevant doses.18
Dendritic spines are highly plastic structures that correlate with changes in synaptic strength. Both stress and exposure to drugs of abuse dynamically mediate spine density within corticolimbic structures, including the nucleus accumbens (NAc), where an increase is typically observed.19,20
Our laboratory demonstrated that sensitization to low-dose KET increases spine density in the NAc shell subregion of males and females, whereas only females had an increase in the NAc core (NAcc).21
NAc spine density alterations following incubation of KET craving are unknown, but incubated cocaine craving results in persistent induction of NAc spine formation over the course of withdrawal,22
associated with increased excitability and thought to mediate the persistence of craving.23
Therefore, we assessed NAcc dendritic spine density following incubation of KET craving in stressed and KET pretreated rats.
In this work, we therefore asked whether or not limited and repeated treatments with low doses of KET enhance vulnerability to KET addiction-like behavior in both sexes of rats.
The purpose of this study was to assess the effects of stress exposure and KET pretreatment in males and females on measures of KET addiction-like behavior and changes in dendritic spine density in the NAcc. KET treatment resulted in a partial amelioration of depression-related behaviors and an increase in thin spine density in the NAcc. CMS enhanced KET addictive-like behavior in females only. Finally, females with prior KET treatment displayed decreased FR1 KET intake. This is the first study to investigate sex differences in KET addictive-like behaviors within the context of stress.
In the current study, first-pass indicators of CMS impact included reduced body weight gain in males and estrous cycle disruptions in females. The lack of body weight gain changes in females was expected and has been previously demonstrated, as females do not gain as much body weight as their age-matched male counterparts.31
Anehdonia, a hallmark of depression, was assessed using the sucrose intake test, where a decrease in consumption of a sweet solution is usaally reduced after CMS.16
In the present study, CMS reduced total sucrose intake in both males and females as compared to same-sex NS counterparts. This is a significant indicator of CMS’s impact on hedonic consummatory behavior and is in line with findings from other species exposed to stressful stimuli.32
Importantly, the decreased intake was still observed after normalizing intake by body weight, suggesting the effect is not due to secondary impacts of CMS.
KET treatment had no effect on sucrose intake; rather, cessation of CMS returned sucrose intake to levels comparable to NS counterparts. Our 21-day CMS protocol was adopted from Li et al.25
, where acute KET (10 mg/kg i.p.) increased sucrose preference in CMS male rats lasting up to 7 days. Similar findings were seen with male mice and sucrose intake.33,34
However, others found no effect of KET in male or female mice on CMS-induced anhedonia,35
and only a transient effect of KET on sucrose preference in socially isolated males, but not females.36
Another depression model, vicarious social defeat,37
produces a depressive-like behavioral profile (including anhedonia) that is reversed by KET in female mice,38
suggesting that stressor type can impact KET’s effectiveness. There are notable methodological differences among these studies that could explain these discrepancies. Drugs delivered via i.p. injection undergo initial absorption in the liver before reaching the brain,39
therefore introducing greater possible effects of drug metabolites. Together, KET’s effects on anhedonia in preclinical studies remain unclear. As these measures were originally developed to test the efficacy of traditional (i.e., monoaminergic) antidepressants predominantly in males, more work is needed to optimize procedures testing rapid-acting antidepressants in females.
The NSFT demonstrated KET’s ameliorating effects on CMS-induced neophagia in both males and females, in line with previous findings.25,26,40
While these studies utilized i.p. injections of acute KET, this study uses i.v. infusions at a clinically relevant dose to reverse CMS-induced behavioral deficits in both sexes.
Rats underwent operant KET self-administration. Due to females’ higher sucrose pellet intake during initial shaping, analysis for males and females was conducted separately. Pharmacokinetic sex differences lend further support for separating the analysis for self-administration by sex, as females have slower clearance and longer elimination half-lives than males.41
Females previously exposed to CMS displayed greater addiction-like behavior than NS females, demonstrated by increased intake during FR1 and enhanced motivation during PR sessions, an effect absent in CMS males. The dose of KET used for self-administration (0.5 mg/kg/inf) is a high dose commonly used.13,15,42,43
However, none of the aforementioned studies included females or tested incubation of craving. This is the first study to demonstrate persistent, incubated craving of KET after 7 to 21 days of forced abstinence. Animals show incubation of craving to many other drugs of abuse with differing temporal profiles.44
In the current study, the expression of craving plateaus between the D7 and D21. It is well known that stress is a potent trigger for drug craving and precipitation of relapse in humans, especially for women.45
Furthermore, female rodents are more susceptible to stress-induced augmentation of addiction-related behavior.46–49
This could be due to a positive interaction between circulating estradiol and extracellular dopamine in the NAc following chronic stress.48
Indeed, behavioral responsivity to KET is influenced by gonadal hormones.50,51
More work is needed to understand the role of gonadal hormones in mediating stress-induced drug-seeking behavior, especially regarding KET.
During FR1 sessions, all animals increased their KET intake over time and maintained those levels after PR testing, indicating robust reinforcement. Among males, there were no differences due to stress conditions or prior KET treatment. Conversely, KET-treated females (regardless of stress condition) reduced their KET intake relative to SAL-treated females after the fifth FR1 session that persisted after PR testing. This suggests that KET pretreatment may have enhanced the motivation to obtain high doses of KET, since their PR and incubation of craving were similar to animals with high intake during FR1. Future studies involving extended access paradigms will help to further characterize this effect.
KET preexposed animals also had increased density of thin spines in the NAcc following incubation of KET craving. Immature spines (thin and stubby subtypes) are more motile and dynamically regulated as compared to the more stable mushroom spines.52
These alterations correlate with increased synaptic excitability, as more synapses form and more glutamate receptors are inserted into the postsynaptic membrane,53
thereby altering subsequent responsivity to drug- or stress-paired stimuli. NAc medium spiny neurons undergo robust structural and physiological changes following exposure to both drugs and chronic stress: in both cases generally, thin and/or stubby dendritic spine densities increase.20,54–56
While our results for males are in accordance with a previous study using socially defeated rats,57
our results in females were unexpected because the last therapeutic KET infusion occurred 40 days prior to analysis. Additionally, another study with CMS males found decreased NAc spine density58
as well as a clinical study showing decreased NAc volume following KET infusion.59
In addition to methodological differences, our effect in females may be an additive result of KET treatment and KET self-administration. Previously, we reported increased spine density in the NAcc only in females, while both sexes had increases in the shell 2 h after an acute KET challenge (i.p.).21
This effect of KET pretreatment on NAcc spines suggests long-lasting neuroadaptations, possibly due to upstream regulation of the medial prefrontal cortex and hippocampus, where KET reverses stress-induced deficits in spineogenesis.60
Although these changes appear to be independent of the expression of KET addiction-like behavior, they may be related to the decreased FR1 intake observed in females and are unlikely to be linked to a specific antidepressant/therapeutic behavioral effect due to lack of specificity to stressed females and males.
Taken together, we have demonstrated a sex-specific responsivity to KET addiction-like behavior following exposure to chronic stress in females, an effect not observed in male counterparts. Additionally, using a novel i.v. KET treatment protocol similar to clinically relevant dosing, we showed that KET treatment partially ameliorates depression-related effects of CMS in both sexes without increasing propensity to self-administer KET as well as increased NAcc dendritic spine density. Finally, KET-treated females displayed decreased KET FR1 intake. Together, these findings suggest a differential response to KET self-administration that was not manifested in the expression of addiction-like behaviors but may still underlie neuroadaptations in reward circuitry relevant to responsivity to stress or drug-related stimuli. While these findings highlight an important question regarding the abuse potential of repeated low-dose therapeutic KET in rats, these factors require investigation in clinical population.