Behavioral and biochemical sensitivity to low doses of ketamine: Influence of estrous cycle in C57BL/6 mice

Abstract

Rationale: Low-dose ketamine is a rapid-acting antidepressant, to which female rodents are more sensitive as compared to males. However, the mechanism mediating this sex difference in ketamine sensitivity remains elusive.

Objectives: We sought to determine whether male and female mice differ in their behavioral sensitivity to low doses of ketamine, and uncover how ovarian hormones influence females' ketamine sensitivity. We also aimed to uncover some of the molecular mechanism(s) in mood-related brain regions that mediate sex differences in ketamine antidepressant effects.

Methods: Male and female mice (freely-cycling, diestrus 1 [D1], proestrus [Pro], or D1 treated with an estrogen receptor (ER) α, ERβ, or progesterone receptor (PR) agonist) received ketamine (0, 1.5, or 3 mg/kg, intraperitoneally) and were tested in the forced swim test (FST) 30 min later. Ketamine's influence over synaptic plasticity markers in the prefrontal cortex (PFC) and hippocampus (HPC) of males, D1, and Pro females was quantified by Western blot 1 h post-treatment.

Results: Males, freely cycling females, D1 and Pro females exhibited antidepressant-like responses to 3 mg/kg ketamine. Pro females were the only group where ketamine exhibited an antidepressant effect at 1.5 mg/kg. D1 females treated with an agonist for ERα or ERβ exhibited an antidepressant-like response to 1.5 mg/kg ketamine. Ketamine (3 mg/kg) increased synaptic plasticity-related proteins in the PFC and HPC of males, D1, and Pro females. Yet, Pro females exhibited an increase in p-Akt and p-CaMKIIα in response to 1.5 and 3 mg/kg ketamine.

Conclusion: Our results indicate that females' enhanced sensitivity to ketamine during Pro is likely mediated through estradiol acting on ERα and ERβ, leading to greater activation of synaptic plasticity-related kinases within the PFC and HPC.

Keywords: Estrogen; Estrous cycle; Forced swim test; Ketamine; Progesterone; Sex differences.

Figure 1. Behavioral sensitivity to the antidepressant-like effect of ketamine is influenced by sex

Ketamine at 3 mg/kg reduced immobility time in males and freely-cycling females. Overall, females exhibited lower immobility time as compared to males. *p<0.05 vs. vehicle, #p<0.05 males vs. females. Data are means + SEM. (males: n=7–8/group, females 8/group).

Figure 2. Behavioral sensitivity to the antidepressant-like effect of ketamine is influenced by sex and by estrous stage

(A) Ketamine at 3 mg/kg reduced immobility time in males and diestrus 1 (D1) females, 1.5 and 3 mg/kg ketamine reduced immobility time in proestrus (Pro) females (*p<.05 vs. within-group vehicle). (B) Representative vaginal lavage samples from D1 and Pro females. (C) Plasma levels of estradiol (pg/mL) and levels of progesterone (ng/mL) were significantly lower in females in D1 vs. Pro females (*p<.05, ** p <.01). Data are means + SEM. Different letters indicate statistically significant differences within the treatment group. (males: n=8/group, D1: n=7–9/group, Pro: n=7–10/group; plasma: n=5–7)

Figure 3. Behavioral sensitivity to the antidepressant-like effect of ketamine is influenced by agonists for ERα and ERβ

Ketamine at 1.5 mg/kg reduced immobility time for D1 females treated with PPT (ERα agonist) or DPN (ERβ agonist), but not progesterone (P4). *p<0.05 vs. within-group vehicle; Different letters indicate statistically significant differences within the treatment group. Data are means + SEM. (n=7–10/group)

Figure 4. Influence of sex/estrous stage on protein expression of synaptic plasticity markers within the prefrontal cortex (PFC) following treatment with vehicle or ketamine

(A) Protein expression of p-CaMKIIα/CaMKIIα was not affected by ketamine nor by sex/estrous stage. (B) Males and Pro females exhibited an increased expression of p-GluR1/GluR1 and (C) increased expression of BDNF following 3 mg/kg ketamine. (D) Protein expression of p-MAPK/MAPK was not affected by ketamine, yet there was a main effect of sex/estrous stage. (E) Ketamine at 1.5 mg/kg increased expression of p-Akt/Akt in Pro females, and 3 mg/kg increased expression in males and Pro females. (F) Ketamine at 1.5 mg/kg increased expression of p-GSK-3β/GSK-3β in males, and in males and Pro females at 3 mg/kg. (G) Ketamine at 3 mg/kg increased p-mTOR/mTOR expression in males and D1 females. *p<.05, **p<.005, ***p<.0005, ****p<0.0001 vs. within-group vehicle. Different letters indicate statistically significant differences between sex/estrous stages within the vehicle treatment group. Data are means + SEM. (n=4–8/group)

Figure 5. Influence of sex/estrous stage on protein expression of synaptic plasticity markers within the hippocampus (HPC) following treatment with vehicle or ketamine

(A) Protein expression of p-CaMKIIα/CaMKIIα was increased by 1.5 mg/kg ketamine in Pro females, and in males and Pro females by 3 mg/kg. (B) Ketamine at 3 mg/kg increased expression of p-GluR1/GluR1 in males and Pro females, (C) and increased expression of BDNF in males and D1 females. (D) Protein expression of p-MAPK/MAPK was significantly lower in D1 females in all treatment conditions. Ketamine at 1.5 mg/kg increased expression of p-MAPK in D1 females, and in all groups at 3 mg/kg. (E) Ketamine at 1.5 mg/kg increased expression of p-Akt/Akt in Pro females, and 3 mg/kg increased expression in males and Pro females. (F) Ketamine at 1.5 mg/kg increased expression of p-GSK-3β/GSK-3β in males, and in all groups at 3 mg/kg. (G) Ketamine at 3 mg/kg increased p-mTOR/mTOR expression in males and D1 females. *p<.05, **p<.005, ***p<.0005, ****p<0.0001 vs. within-group vehicle. Different letters indicate statistically significant differences between sex/estrous stages within the vehicle treatment group. Data are means + SEM. (n=4–7/group)

Figure 6. Schematic representation of the hypothesized shared influence of ketamine and ovarian hormones within mood related brain regions

(A) A simplified schematic of the intracellular signaling pathways that are influenced by ketamine and ovarian hormones within the PFC and (B) and the HPC. Yellow circles indicate pathways activated by behaviorally-effective doses of ketamine in Pro females.