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

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.

Sex- and estrous-cycle dependent dorsal hippocampal phosphoproteomic changes induced by low-dose ketamine.

Numerous emotional and cognitive processes mediated by the hippocampus present differences between sexes and can be markedly influenced by hormonal status in males and females of several species. In rodents, the dorsal hippocampus (dHPC) is known to contribute to the rapid antidepressant actions of the NMDA receptor antagonist ketamine. We and others have demonstrated a greater sensitivity to the fast-acting antidepressant ketamine in female versus male rats that is estrogen- and progesterone-dependent. However, the underlying mechanisms remain unclear. Using an acute low dose (2.5 mg/kg) of ketamine that is behaviorally effective in female but not male rats, a label-free phosphoproteomics approach was employed to identify ketamine-induced changes in signaling pathway activation and phosphoprotein abundance within the dHPC of intact adult male rats and female rats in either diestrus or proestrus. At baseline, males and females showed striking dissimilarities in the dHPC proteome and phosphoproteome related to synaptic signaling and mitochondrial function-differences also strongly influenced by cycle stage in female rats. Notably, phosphoproteins enriched in PKA signaling emerged as being both significantly sex-dependent at baseline and also the primary target of ketamine-induced protein phosphorylation selectively in female rats, regardless of cycle stage. Reduced phosphoprotein abundance within this pathway was observed in males, suggesting bi-directional effects of low-dose ketamine between sexes. These findings present biological sex and hormonal milieu as critical modulators of ketamine's rapid actions within this brain region and provide greater insight into potential translational and post-translational processes underlying sex- and hormone-dependent modulation of ketamine's therapeutic effects.

Sex Differences in the Pharmacokinetics of Low-dose Ketamine in Plasma and Brain of Male and Female Rats.

Recent work from our group and others has revealed a higher sensitivity of female rodents to the antidepressant-like effects of the N-methyl d-aspartate receptor antagonist ketamine strongly influenced by circulating estrogen and progesterone levels. However, in the absence of any preclinical studies of pharmacokinetic sex differences using low-dose ketamine in rats, it is unclear whether the effects of sex and hormonal milieu on ketamine's behavioral actions are influenced by differences in ketamine metabolism between male and female rats. Therefore, this work examined whether sex and hormonal status affect ketamine metabolism and distribution in male and female rats using a low antidepressant-like dose selectively effective in females. Intact male rats and female rats in either diestrus (low estrogen, progesterone) or proestrus (high estrogen, progesterone) were administered low-dose ketamine, and their plasma and brains were collected to analyze levels of ketamine and its metabolites norketamine (NK) and dehydronorketamine. Females exhibited greater concentrations of ketamine and NK over the first 30 min following treatment in both brain and plasma, largely accounted for by slower clearance rates and longer half-lives. Interestingly, despite the impact of ovarian hormones on behavioral sensitivity to ketamine, no appreciable differences in pharmacokinetic parameters existed between proestrus and diestrus female rats. This work is the first to demonstrate sex differences in ketamine pharmacokinetics in rats, and suggests that while sex differences in metabolism may influence the amount of ketamine and NK reaching target areas in the brain, the impact of circulating hormone levels here is negligible.

Hedonic sensitivity to low-dose ketamine is modulated by gonadal hormones in a sex-dependent manner.

We recently reported a greater sensitivity of female rats to rapid antidepressant-like effects of ketamine compared to male rats, and that ovarian-derived estradiol (E2) and progesterone (P4) are essential for this response. However, to what extent testosterone may also contribute, and whether duration of response to ketamine is modulated in a sex- and hormone-dependent manner remains unclear. To explore this, we systematically investigated the influence of testosterone, estradiol and progesterone on initiation and maintenance of hedonic response to low-dose ketamine (2.5 mg/kg) in intact and gonadectomized male and female rats. Ketamine induced a sustained increase in sucrose preference of female, but not male, rats in an E2P4-dependent manner. Whereas testosterone failed to alter male treatment response, concurrent administration of P4 alone in intact males enhanced hedonic response low-dose ketamine. Treatment responsiveness in female rats only was associated with greater hippocampal BDNF levels, but not activation of key downstream signaling effectors. We provide novel evidence supporting activational roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to low-dose ketamine in female and male rats, respectively. Organizational differences may, in part, account for the persistence of sex differences following gonadectomy and selective involvement of BDNF in treatment response.

The Anxiolytic and Antidepressant-like Effects of Testosterone and Estrogen in Gonadectomized Male Rats.

BACKGROUND: While the influence of testosterone levels on vulnerability to affective disorders is not straightforward, research suggests this hormone may confer some degree of resiliency in men. We recently demonstrated a role for the dentate gyrus in mediating testosterone's protective effects on depressive-like behavior in gonadectomized male rats. Here, testosterone may exert its effects through androgen receptor-mediated mechanisms or via local aromatization to estradiol. METHODS: Gonadectomized male rats were implanted with a placebo, testosterone, or estradiol pellet, and subsequent protective anxiolytic- and antidepressant-like effects of testosterone and its aromatized metabolite, estradiol, were then investigated in the open field and sucrose preference tests, respectively. Moreover, their influence on gene expression in the hippocampus was analyzed by genome-wide complementary DNA microarray analysis. Finally, the contribution of testosterone's aromatization within the dentate gyrus was assessed by local infusion of the aromatase inhibitor fadrozole, whose efficacy was confirmed by liquid chromatography-tandem mass spectrometry. RESULTS: Both hormones had antidepressant-like effects associated with a substantial overlap in transcriptional regulation, particularly in synaptic plasticity- and mitogen-activated protein kinase pathway-related genes. Further, chronic aromatase inhibition within the dentate gyrus blocked the protective effects of testosterone. CONCLUSIONS: Both testosterone and estradiol exhibit anxiolytic- and antidepressant-like effects in gonadectomized male rats, while similarly regulating critical mediators of these behaviors, suggesting common underlying mechanisms. Accordingly, we demonstrated that testosterone's protective effects are mediated, in part, by its aromatization in the dentate gyrus. These findings thus provide further insight into a role for estradiol in mediating the protective anxiolytic- and antidepressant-like effects of testosterone.