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

Abstract

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.

Figure 1. Timeline of procedures and cyclic hormone treatment regimen for Experiment 1.

Abbreviations: E2, 17β-estradiol benzoate; KET, ketamine (2.5 mg/kg); OIL, sesame oil; OVX, ovariectomy; P4, progesterone; SAL, saline.

Figure 2. Estradiol and progesterone are required for rapid and sustained hedonic-like effects of low-dose ketamine in female rats.

(a) Ketamine (KET, 2.5 mg/kg, i.p.) induced a significant and protracted increase in sucrose preference in ovariectomized (OVX) female rats treated with E2P4 (****p < 0.0001, ***p < 0.001, **p < 0.01 vs. SAL), but not OIL, E2 or P4 alone (Main Effects: Treatment/Day, ****p < 0.0001; Hormone: ^ߤp = 0.0693). Data are expressed as mean ± SEM (n = 48). (b–e) Saline (SAL) baseline sucrose preference levels predicted magnitude of positive response to KET in E2P4-treated OVX females only (r² = 0.7035, p = 0.0007). (f) Significantly reduced overall body weight gain of E2-treated OVX female rats compared to OVX + OIL females (***P < 0.001) confirmed efficacy of hormone treatment. Data are expressed as mean ± SEM (n = 48).

Figure 3. Cyclic P4 treatment enhances the hedonic sensitivity of intact male rats to low-dose ketamine.

(a) Cyclic P4 treatment increased sensitivity of male rats to low-dose ketamine (KET) treatment (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs. SAL) for up to 6 days following drug administration (Main Effects: Treatment/Day, ***p < 0.001; Hormone: ***p < 0.001). Data are expressed as mean ± SEM (n = 40). (b–e) Lower saline (SAL) baseline sucrose preference levels predicted a higher magnitude of positive response to KET in treatment-responsive P4-treated intact males (r² = 0.9692, P < 0.0001). (f) Cyclic treatment with E2 alone or in combination with P4 significantly reduced overall body weight gain of intact male rats compared to Intact + OIL males (***P < 0.001), confirming effective hormone treatment. Data are expressed as mean ± SEM (n = 38).

Figure 4. Chronic testosterone treatment blocks pro-hedonic like effects of ketamine in intact female rats via persistent disruption of estrous cyclicity.

(a) Chronic testosterone treatment in intact female rats induced anhedonic behavior (**P < 0.005) and blocked response to ketamine (2.5 mg/kg, i.p.) (P > 0.05). Acute ketamine treatment led to a modest increase in sucrose preferences of placebo-treated intact female rats (**P < 0.01, *P < 0.05 vs. SAL) that persisted for 5 days (Main Effects: Treatment/Day, **P < 0.01; Hormone: *P < 0.05). Data are expressed as mean ± SEM (n = 18). (b,c) Lower SAL baseline sucrose preference levels predicted a higher magnitude of positive response to KET in treatment-responsive placebo-treated intact females (r² = 0.8463, P = 0.0002), but not those receiving testosterone pellets (P > 0.05). (d) Chronic treatment with testosterone significantly increased overall body weight gain of intact female rats relative to intact females receiving placebo pellets (**P < 0.01). Data are expressed as mean ± SEM (n = 18). (e) Chronic testosterone treatment resulted in persistent disruption of estrous cyclicity in intact female rats.

Figure 5. Gonadal testosterone does not influence sensitivity to low-dose ketamine in male rats.

(a) Gonadectomized (GDX) male rats displayed a significantly lower sucrose preference when compared to SHAM & testosterone-supplemented male rats (**P < 0.01). Ketamine (KET; 2.5 mg/kg, i.p.) was without effect in male rats, regardless of hormonal status (Main Effects: Treatment/Day, ***P < 0.001; Hormone: **P < 0.01). Data are expressed as mean ± SEM (n = 30). (b–d) Saline (SAL) baseline preference levels of all males were not associated with magnitude of response to KET (P > 0.05). (e) Gonadal testosterone depletion resulted in significantly less body weight gain throughout the experiment relative to SHAM-operated males (*P < 0.05). Chronic testosterone supplementation at the time of gonadectomy was sufficient to block this effect (P > 0.05). Data are expressed as mean ± SEM (n = 30).

Figure 6. Integrated analysis of ketamine’s effects across sex and hormonal status.

(a) Comparison of all groups of ovariectomized (OVX) and intact female rats demonstrating that normal cyclic fluctuation of both estradiol (E2) and progesterone (P4) levels are essential for pro-hedonic like response to low-dose ketamine (KET; 2.5 mg/kg) (*P < 0.05 vs. OVX + OIL). Data are expressed as mean ± SEM (n = 66). (b) Comparison of all groups of gonadectomized (GDX) and intact male rats reiterate the negligible effect of circulating testosterone (T) levels on male sensitivity to low-dose KET (P > 0.05), but identify effective enhancement of KET sensitivity in intact male rats by P4 treatment (*P < 0.05 vs. Intact + OIL). Data in (a,b) are presented as the percent change in sucrose preference following KET administration relative to saline (SAL) baseline levels, averaged across all days of the post-treatment period. Data are expressed as mean ± SEM (n = 68). (c,d) Standardization of female (n = 66) and male (n = 68) sucrose preference scores presented in (a,b), respectively, via Z-score transformation relative to OIL-treated groups of each sex (**P < 0.01, *P < 0.05). Data are expressed as mean ± SEM. (e) Percent change in sucrose preference levels from baseline following KET administration compared across sex and all hormone treatments via Z-score normalization of each group’s scores to OVX + OIL female rats. The magnitude of pro-hedonic like effects of KET was found to be similar in OVX + E2P4 (**P < 0.01) and Intact + P (*P < 0.05) female and P4-treated intact males (*P < 0.05) when controlling for unequal variances between all experimental cohorts. Data are expressed as mean ± SEM (n = 134).

Figure 7. Protein levels of BDNF and downstream signaling effectors 24 h after ketamine in estradiol- and progesterone-treated female and male rats.

(a) Hippocampal BDNF levels were significantly increased 24 h after low-dose ketamine (2.5 mg/kg, i.p.) only in ovariectomized female rats receiving cyclic treatment with both estradiol and progesterone (E2P4) relative to OIL-treated controls (*p = 0.0345; all other p > 0.05). (b) BDNF in the hippocampus of intact male rats was decreased 24 h after ketamine in estradiol (E2; ***p = 0.0006) and E2P4-treated (**p = 0.0034) male rats relative to OIL-treated controls, but was unaffected in those receiving progesterone (P4) alone (p > 0.05). (c,d) Neither total nor phosphorylated levels of hippocampal AKT were altered 24 h post-ketamine ketamine in male and female rats regardless of hormone treatment. (e,f) Phosphorylated ERK1/2 levels were increased following ketamine in E2-treated females (**p = 0.0047), but were otherwise unaffected (p > 0.05) in all other treatment conditions. Total ERK abundance was similar between groups, except in P4-treated males which displayed decreased ERK relative to OIL-treated controls (*p = 0.0338). (g,h) While CaMKIIα phosphorylation was not associated with treatment-response in either sex, lower levels were apparent in E2- (****p < 0.0001) and E2P4-treated (****p < 0.0001) male rats 24 h following ketamine. Parallel decreases in total CaMKIIα levels were observed in the same males relative to OIL-treated counterparts (E2: *p = 0.0196; E2P4: **p = 0.0017). Lower CaMKIIα abundance was also observed at this timepoint in E2- (**p = 0.0033) and P4-treated (****p < 0.0001)—but not E2P4-treated—female rats when compared to same-sex controls. (i) Representative western blots for proteins depicted in (c,h) across all treatment groups. Vertical lines indicate juxtaposition of non-adjacent regions within the same membrane for each phosphorylated and total protein assayed. All data expressed as mean ± SEM (n = 24 female/24 male).