Estradiol replacement extends the window of opportunity for hippocampal function

Abstract

We previously reported that treating aged female rats, ovariectomized (OVX) as young adults, with acute proestrous levels of 17β estradiol (E2) increases CA1 spine density, NMDAR to AMPAR ratio, GluN2B-mediated NMDAR current, and long-term potentiation at CA3-CA1 synapses if administered by 15, but not at 19-month post-OVX, defining the critical window of opportunity. Importantly, when rats are aged with ovaries intact until OVX at 20 months, hippocampal E2 responsiveness is maintained, indicating the deficit at 19-month post-OVX is a consequence of the duration of hormone deprivation and not chronological age. Here, we find the beneficial effect of E2 on novel object recognition in OVX rats was constrained by the same critical window. Furthermore, chronic low-level E2 replacement, commenced by 11-month post-OVX using subcutaneous capsules removed 2 weeks before acute proestrous E2 treatment, prevents the loss of hippocampal responsiveness at 19-month post-OVX. These data define the dynamic nature of the critical window showing that chronic replacement with physiological E2 levels within a certain period post-OVX can lengthen the window.

Keywords: 17ß-estradiol; E2; GluN2B; Learning and memory; Long term potentiation; NMDA; NR2B; Plasticity.

Fig. 1. Timeline and procedures for experimental paradigms

A. 9 month post-OVX NOR and Adult-Replaced experiments. B. 15 month post-OVX NOR and Middle-Age Replaced experiments. C. 19 month post-OVX NOR. D. Aged ovary intact NOR.

Fig. 2. E2-induced NOR is lost after 19-mo post-OVX and this loss is not due to chronological age alone

A1–C1. During training for all groups, the percent time spent on the right object was not different than chance (50%), indicating no side preference. The percent time spent on the novel object during testing was significantly increased compared to chance with acute proestrous-like E2 treatment in 9-month post-OVX (A2) and 15-month post-OVX (B2) but not in 19-month post-OVX (C2) rats. D. Rats aged with ovaries intact and undergo OVX at 20 months of age have significant NOR when treated with acute proestrous-like E2 at the same chronological age as 19-month post-OVX rats. No significant NOR occurred for the Vehicle-treated rats for any of the experimental groups. + p<0.05 compared to chance. Arrows on the right indicate the direction of change in LTP magnitude at CA3-CA1 synapses in E2 versus V-treated OVX rats at these time points previously published in Smith et al, 2010.

Fig. 3. Chronic low-level E2-replacement beginning at 9.5–11 months post-OVX protects against the loss of the beneficial effects of acute proestrous-like E2 treatment on NOR and synaptic function, but not on dendritic spine density

A. Plot showing a significant increase in the average LTP at CA3-CA1 synapses recorded in slices at 19 months post-OVX from rats chronically treated with E2 compared to Chol for 7–8.5 months (Adult-Replaced), then subsequently treated with acute proestrous-like E2 and sacrificed 24 hrs following the second E2 injection. Inset shows average percent LTP with each animal in the dataset presented as an individual closed circle. Representative waveforms for Chol and E2 replaced rats with dotted line representing baseline and solid line representing 30 min post-tetanus. Scale bar represents 0.3 mV on y-axis and 50ms on x-axis. B. Representative whole-cell recording from a Chol-replaced rat. Plot shows change in glutamatergic current with sequential pharmacological blockade of GluN2B-containing NMDARs using Ro 25-6981 (1 μM), NMDARs using D-L, APV (100 μM), and AMPARs using DNQX (10 μM). Waveforms represent total glutamate, AMPAR, NMDAR, and GluN2B currents. Scale bar represents 100pA on y-axis and 300ms on x-axis. C. Bar chart shows a significant increase in the NMDAR/AMPAR ratio recorded in CA1 pyramidal cells from E2- versus Chol-replaced rats. D. Bar chart shows a significant increase in the GluN2B/total NMDAR ratio in CA1 pyramidal cells recorded from E2- versus Chol-replaced rats. E. Example images of dendritic spines from Chol and E2-replaced rats. F. Bar chart shows no difference in number of CA1 dendritic spines on tertiary dendrites per 10μm from Chol and E2-replaced rats. G. Bar chart shows significant NOR only in chronic E2-replaced rats. * indicates p<0.05, + p<0.05 compared to chance.

Fig. 4. Chronic low-level E2-replacement beginning at 15 months post-OVX enables NOR in the absence of preserving the beneficial effects of acute proestrous-like E2 treatment on synaptic function and spine density

A. Plot showing no difference in the average LTP magnitude at CA3-CA1 synapses recorded in slices at 19 months post-OVX from rats chronically treated with Chol or E2 for 3 months (Middle-Age-Replaced), then subsequently treated with acute proestrous-like E2 and sacrificed 24 hrs following the second E2 injection. Inset shows average percent LTP with each animal in the dataset presented as an individual closed circle. Representative waveforms for Chol and E2 replaced rats with dotted line representing baseline and solid line representing 30 min post-tetanus. Scale bar represents 0.3 mV on y-axis and 50ms on x-axis. B. Representative whole-cell recording from a Chol-replaced rat. Plot shows change in glutamatergic current with sequential pharmacological blockade of GluN2B-containing NMDARs using Ro 25-6981 (1 μM), NMDARs using D-L, APV (100 μM), and AMPARs using DNQX (10 μM). Waveforms represent total glutamate, AMPAR, NMDAR, and GluN2B currents. Scale bar represents 100pA on y-axis and 300ms on x-axis. C. Bar chart shows no difference in the NMDAR/AMPAR ratio in CA1 pyramidal cells recorded from Chol versus E2-replaced rats. D. Bar chart shows no significant difference in the GluN2B/total NMDAR ratio recorded from CA1 pyramidal cells recorded from Chol versus E2-replaced rats. E. Example images of dendritic spines from Chol and E2-replaced rats. F. Bar chart shows no difference in the number of dendritic spines on tertiary dendrites per 10μm between Chol and E2-replaced rats. G. Bar chart shows significant NOR only in chronic E2-replaced rats. * indicates p<0.05, + p<0.05 compared to chance.