Cyclodextrin-encapsulated 17β-estradiol (E2; Sigma, St. Louis, MO) at a dose of 0.2 mg/kg was dissolved in physiological saline in a volume of 4 ml/kg, and injected intraperitoneally (i.p.). This dose in mice facilitates object memory consolidation in the task used here (Gresack and Frick, 2004 (link), 2006 (link)). The vehicle, hydroxypropyl-β-cyclodextrin (HBC), was dissolved in an equal volume of saline and contained the same amount of cyclodextrin as E2. The MEK inhibitor SL327 (α-[Amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile; Sigma), at a dose of 30 mg/kg, was dissolved in 100% dimethyl sulfoxide (DMSO) and injected i.p. in a volume of 2.0 ml/kg. Vehicle controls received HBC or both HBC and DMSO. For intrahippocampal (IH) infusions, physiological saline or cyclodextrin-encapsulated E2 dissolved in physiological saline (5.0 μg/0.5μl) was infused at 0.5 μl/min for 1 minute.
To demonstrate that E2-induced increases in object recognition were dependent on dorsal hippocampal ERK activation, other mice received IH infusions of vehicle or the MEK inhibitor U0126 (1,4-Diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene; 2.0 μg/μl; Sigma) concurrently with i.p. E2 injection or intracerebroventricular (ICV) infusion of bovine serum albumin-conjugated 17β-estradiol (see below) into the dorsal third ventricle. U0126 was dissolved in 100% DMSO to 4 μg/μl as a stock solution and then serially diluted in physiological saline for infusion of various doses. U0126 or vehicle (50% DMSO) were infused at a rate of 0.50 μl/min and a volume of 0.50 μl/side. Other mice received ICV infusions of vehicle or bovine serum albumin-conjugated 17β-estradiol (β-Estradiol 6-(O-carboxy-methyl)oxime; BSA-E2; Sigma). The covalent conjugation of E2 to the large BSA molecule prevents E2 from passing through the cell membrane and binding to intracellular ERs (Taguchi et al., 2004 (link)). Thus, effects of BSA-E2 should be mediated by membrane-bound ERs. BSA-E2 was dissolved in Tris-HCl to a concentration of 5.0 μM. Either 5.0 μM BSA-E2 or vehicle (Tris-HCl) was infused at a rate of 0.5 μl/min at a volume of 1.0 μl.
To demonstrate that the effects of BSA-E2 on memory and ERK activation were independent of nuclear estrogen receptors, other mice received ICV infusions of BSA-E2 conducted as described above concurrently with IH infusions of the nuclear estrogen receptor antagonist ICI 182,780 ((7a,17b)-7-[9[(4,4,5,5,5pentafluoropentyl) Sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol; 10 μg/μl; Sigma). ICI 182,780 is an antagonist of ERα and ERβ that impairs E2-induced ER dimerization (Weatherman et al., 2002 (link)) and translocation of ERs into the cell nucleus (Dauvois et al., 1993 (link)). If ICI 182,780 does not block the effects of BSA-E2 on memory and ERK activation, then this would indicate that these effects are mediated by membrane-associated estrogen receptors rather than nuclear receptors. Intrahippocampal infusions of ICI 182,780 were conducted at a rate of 0.5 μl/min and a volume of 0.5 μl/side, resulting in a dose of 5.0 μg/side. In other mice, ICI 182,780 was infused intrahippocampally without concurrent ICV infusion as a control due to a report that this compound in female rats can act as an estrogen receptor agonist to enhance place learning when administered in the absence of E2 (Zurkovsky et al., 2006 (link)). To compare effects of ICI 182,780 with those of traditional E2, 0.2 mg/kg E2 was administered i.p. either alone or with ICI 182,780 infused intrahippocampally. The E2 + IH ICI 182,780 group also served as a control for the effectiveness of the ICI compound in blocking the effects of traditional, non-BSA conjugated E2 on object memory consolidation and ERK activation. Vehicle controls for the aforementioned groups received ICV and IH infusions of saline or BSA dissolved in saline.
Finally, to demonstrate that the effects of BSA-E2 on memory and ERK activation involved the dorsal hippocampus, BSA-E2 was infused bilaterally into the dorsal hippocampus of another set of mice. Intrahippocampal infusions were conducted at a rate of 0.5 μl/min and at a volume of 0.5 μl/side, resulting in doses of 5.0 μM/side. As a control, these mice also received ICV infusions of vehicle (BSA dissolved in saline). Additional mice received ICV BSA-E2 + IH vehicle as a control to replicate the effects of ICV BSA-E2 observed above. As an additional method of demonstrating hippocampal involvement in the BSA-E2 effect, other mice received ICV infusions of BSA-E2 conducted as described above concurrently with IH infusions of the GABAA receptor agonist muscimol (3-Hydroxy-5-aminomethyl-isoxazole; 0.50 μg/μl dissolved in saline; Sigma). Muscimol temporarily inactivates a brain region of interest by increasing GABAergic inhibition without permanently damaging the tissue (Martin, 1991 (link)). Therefore, if muscimol interferes with the beneficial effects of ICV BSA-E2 on memory, then this would suggest critical involvement of the dorsal hippocampus in this effect. Intrahippocampal infusions of muscimol were conducted at the same rate and volume as above. Vehicle controls for all of the aforementioned groups received ICV and IH infusions of saline or BSA dissolved in saline.
Injection cannulae for IH and ICV infusions remained in place for 1 minute after infusion to prevent drug diffusion up the injection track. For behaviorally tested mice, all solutions were administered immediately after the sample phase of object recognition training.
To demonstrate that E2-induced increases in object recognition were dependent on dorsal hippocampal ERK activation, other mice received IH infusions of vehicle or the MEK inhibitor U0126 (1,4-Diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene; 2.0 μg/μl; Sigma) concurrently with i.p. E2 injection or intracerebroventricular (ICV) infusion of bovine serum albumin-conjugated 17β-estradiol (see below) into the dorsal third ventricle. U0126 was dissolved in 100% DMSO to 4 μg/μl as a stock solution and then serially diluted in physiological saline for infusion of various doses. U0126 or vehicle (50% DMSO) were infused at a rate of 0.50 μl/min and a volume of 0.50 μl/side. Other mice received ICV infusions of vehicle or bovine serum albumin-conjugated 17β-estradiol (β-Estradiol 6-(O-carboxy-methyl)oxime; BSA-E2; Sigma). The covalent conjugation of E2 to the large BSA molecule prevents E2 from passing through the cell membrane and binding to intracellular ERs (Taguchi et al., 2004 (link)). Thus, effects of BSA-E2 should be mediated by membrane-bound ERs. BSA-E2 was dissolved in Tris-HCl to a concentration of 5.0 μM. Either 5.0 μM BSA-E2 or vehicle (Tris-HCl) was infused at a rate of 0.5 μl/min at a volume of 1.0 μl.
To demonstrate that the effects of BSA-E2 on memory and ERK activation were independent of nuclear estrogen receptors, other mice received ICV infusions of BSA-E2 conducted as described above concurrently with IH infusions of the nuclear estrogen receptor antagonist ICI 182,780 ((7a,17b)-7-[9[(4,4,5,5,5pentafluoropentyl) Sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol; 10 μg/μl; Sigma). ICI 182,780 is an antagonist of ERα and ERβ that impairs E2-induced ER dimerization (Weatherman et al., 2002 (link)) and translocation of ERs into the cell nucleus (Dauvois et al., 1993 (link)). If ICI 182,780 does not block the effects of BSA-E2 on memory and ERK activation, then this would indicate that these effects are mediated by membrane-associated estrogen receptors rather than nuclear receptors. Intrahippocampal infusions of ICI 182,780 were conducted at a rate of 0.5 μl/min and a volume of 0.5 μl/side, resulting in a dose of 5.0 μg/side. In other mice, ICI 182,780 was infused intrahippocampally without concurrent ICV infusion as a control due to a report that this compound in female rats can act as an estrogen receptor agonist to enhance place learning when administered in the absence of E2 (Zurkovsky et al., 2006 (link)). To compare effects of ICI 182,780 with those of traditional E2, 0.2 mg/kg E2 was administered i.p. either alone or with ICI 182,780 infused intrahippocampally. The E2 + IH ICI 182,780 group also served as a control for the effectiveness of the ICI compound in blocking the effects of traditional, non-BSA conjugated E2 on object memory consolidation and ERK activation. Vehicle controls for the aforementioned groups received ICV and IH infusions of saline or BSA dissolved in saline.
Finally, to demonstrate that the effects of BSA-E2 on memory and ERK activation involved the dorsal hippocampus, BSA-E2 was infused bilaterally into the dorsal hippocampus of another set of mice. Intrahippocampal infusions were conducted at a rate of 0.5 μl/min and at a volume of 0.5 μl/side, resulting in doses of 5.0 μM/side. As a control, these mice also received ICV infusions of vehicle (BSA dissolved in saline). Additional mice received ICV BSA-E2 + IH vehicle as a control to replicate the effects of ICV BSA-E2 observed above. As an additional method of demonstrating hippocampal involvement in the BSA-E2 effect, other mice received ICV infusions of BSA-E2 conducted as described above concurrently with IH infusions of the GABAA receptor agonist muscimol (3-Hydroxy-5-aminomethyl-isoxazole; 0.50 μg/μl dissolved in saline; Sigma). Muscimol temporarily inactivates a brain region of interest by increasing GABAergic inhibition without permanently damaging the tissue (Martin, 1991 (link)). Therefore, if muscimol interferes with the beneficial effects of ICV BSA-E2 on memory, then this would suggest critical involvement of the dorsal hippocampus in this effect. Intrahippocampal infusions of muscimol were conducted at the same rate and volume as above. Vehicle controls for all of the aforementioned groups received ICV and IH infusions of saline or BSA dissolved in saline.
Injection cannulae for IH and ICV infusions remained in place for 1 minute after infusion to prevent drug diffusion up the injection track. For behaviorally tested mice, all solutions were administered immediately after the sample phase of object recognition training.