Subjects were 20 cognitively normal post-menopausal women (See for demographic information). Four additional subjects passed the screening but withdrew before beginning hormone treatment because of the time commitment of the study. Subjects were randomly assigned to receive either three months of 1.0 mg oral 17-β estradiol (E2) per day or placebo. Demographic characteristics including age did not differ between the two groups (See ).
Demographic data (means and standard deviations) for the estradiol and placebo treatment groups. No group differences were found in any of the demographic variables presented (smallest p: p > .19 for BMI).
Subjects were recruited through notices and advertisements in local newspapers and direct mailings. Subjects were required to be postmenopausal and without surgically-induced menopause. Exclusion criteria included use of tobacco or nicotine products within the last five years, a history of breast cancer, and use of hormone therapy during the last year. Twelve subjects had previously taken hormone or estrogen therapy after menopause. The length of time of prior hormone use ranged from one week to 15 years (M = 7.01, SD = 6.7; See ). There was no difference between the estrogen and placebo treatment groups on length of prior estrogen use. Medical exclusion criteria for E2 treatment included: contraindications for hormone therapy, estrogen-dependent neoplasia, untreated blood pressure greater than 160/100, history of deep vein thrombosis or other thromboembolic disease, hepatoma, severe migraines or stroke on oral contraceptives, current use of barbiturates, rifampin, insulin, carbamezepine, oral hypoglycemics, antidepressants, or lipid-lowering drugs, known intolerance to conjugated estrogens, diabetes, untreated thyroid disease, clinical osteoporosis, and a history or presence of severe menopausal symptoms. In addition, we also excluded women with a history of the following: heavy alcohol (more than an average of 1 drink per day) or coffee use (more than three cups per day), significant cardiovascular disease, asthma, active peptic ulcer, hyperthyroidism, pyloric stenosis, narrow angle glaucoma, epilepsy, or current Axis I psychiatric disorders. The alcohol criterion was used to ensure subjects were not alcohol abusing, and the caffeine criterion was used to ensure subjects would not experience caffeine withdrawal on testing days.
Upon meeting these criteria, subjects were approved for further screening at the University of Vermont (UVM) General Clinical Research Center (GCRC). After signing informed consent documents, subjects gave a medical history, underwent a physical and laboratory tests assessing hematopoietic, renal, hepatic and hormonal function. Subjects were cognitively evaluated using the Mini Mental State Exam (MMSE; Folstein et al. 1975
), Brief Cognitive Rating Scale (Reisberg et al. 1988
), and the Mattis Dementia Rating Scale (DRS; Jurica et al. 2001
) to establish a Global Deterioration Scale score (GDS) which rated the degree of cognitive impairment (Reisberg and Ferris 1988
). Subjects were required to have an MMSE score greater than or equal to 27, a DRS score of 123 or greater, and a GDS score of 1 or 2.
Behavioral screening consisted of a partial Structured Clinical Interview for DSM-IV-TR (SCID; First et al. 2001
) to establish the presence/absence of Axis I psychiatric disorders. In addition, subjects completed the Beck Depression Inventory (BDI). A cut off score of 10 was used for the BDI, and subjects scoring over this criterion were discontinued from further participation. All subjects met these criteria for the cognitive and behavioral screening.
After meeting all inclusion criteria, subjects were randomly and blindly assigned to the E2 or placebo condition for three months. In the E2 condition, subjects took 1 mg of oral 17-beta E2 per day for three months. In the placebo condition subjects took similar appearing placebo pills for three months. After three months of treatment estradiol levels were greater for the estradiol group, M = 51.0 pg/ml (SD = 24.4), compared to the placebo group, M = 4.70 pg/ml (SD = 3.8; t(28) = 5.97, p <.001). After three months, subjects completed three cholinergic challenge days (described below). After completion of the challenge days which took approximately three weeks, all subjects took 10 mg per day of medroxyprogesterone acetate for 12 days to produce sloughing of any endometrium that developed.
After three months of estradiol or placebo treatment, subjects came to the UVM GCRC for three drug challenge days. These procedures are detailed in several prior papers and are not repeated here (see (Dumas et al. 2006
; Dumas et al. 2008a
; Dumas et al. 2008b
). The current study examined brain activation at baseline and during the placebo drug challenge day in order to assess the effects of estradiol treatment alone on brain activation. The challenge days were separated by a minimum of forty-eight hours to ensure complete washout of any study medication as was done in our prior studies. Two hours into the study day the fMRI session began with the visual verbal N-back.
fMRI Working Memory Task
We used a visually presented verbal N-back sequential letter task to probe working memory circuitry, wherein subjects saw a string of consonants (except L, W, and Y), presented in upper case letters, one every 3 seconds. Four conditions were presented: 0-back, 1-back, 2-back, and 3-back and on every trial subjects had to make a decision whether the current letter matched according to the rule which changed between blocks. Subjects completed 3 blocks of each condition that occurred in a pseudo-random order such that the same condition did not occur two times in row. Each block contained nine trials.
The 0-back control condition had a minimal working memory load; subjects were asked to decide if the current letter matched a single target letter that was specified before the block began. In the 1-back condition, they were asked to decide if the current letter matched the previous letter. During the 2-back condition, the task was to decide whether the letter currently presented matched the letter that had been presented two back in the sequence; the more difficult 3-back condition required subjects to decide if the current letter matched the letter three back in the sequence. Subjects responded to all items by button press through an MRI compatible fiber optic button response system (Eloquence System, Invivo Corp., Gainesville, FL) to indicate whether the item matched the target condition. Stimuli were delivered through an MR-safe computer monitor. Experimental tasks were presented by PC interface and were programmed using the E-Prime software package; the PC recorded subject responses and reaction times. The task ran for 8 minutes and 12 seconds.
fMRI Scan Procedure and Preprocessing
For logistical reasons, the first 11 subjects were scanned on one magnet while the last nine subjects were tested on a different magnet. The magnets were both Philips 3.0 Tesla Achieva scanners, all procedures and protocol files were the same on each magnet, and the same stimulus delivery and response equipment was used throughout the whole study. A comparison of the 0-back control conditions for subjects scanned on the two different magnets at baseline showed only small differences in the posterior cingulate. As will be shown below, no differences were seen in brain regions responsive to estradiol treatment. Thus, differences between different magnets do not explain the data pattern described below.
The MRI procedures were as follows. All subjects received the following MR sequences as part of the imaging protocol: (1) A sagittal T1-weighted spoiled gradient volumetric sequence oriented perpendicular to the anterior commissure (AC)-posterior commissure (PC) line using a repetition time (TR) of 9.9 ms, echo time (TE) of 4.6 ms, a flip angle of 8 degrees, number signal averages (NSA) 1.0, a field of view (FOV) of 256 mm, a 256 X 256 matrix, and 1.0 mm slice thickness with no gap for 140 contiguous slices. (2) An axial T2-weighted gradient spin echo (GRASE) sequence using the AC-PC line for slice positioning. Twenty eight contiguous slices of 5 mm slice thickness and no gap were acquired using TR 2466 ms, TE 80 ms, NSA 3.0 and FOV of 230 mm. All images were reviewed by a board-certified neuroradiologist to exclude intracranial pathology. fMRI was performed using EpiBOLD (echoplanar blood oxygenation level dependent) imaging. For the fMRI sequences, a single-shot, gradient-echo, echoplanar pulse sequence was used (TR 2500 ms/TE 35 ms/flip angle 90 degrees/1 NSA). Resolution was 2.5 mm × 2.8 mm × 5.0 mm. Thirty contiguous slices of 5mm thickness with no gap were obtained in the axial oblique plane, parallel to the AC-PC line using a FOV of 240 mm and a matrix size of 128 × 96. Field map correction for magnetic inhomogeneities was accomplished by acquiring images with offset TE at the end of the functional series.
Preprocessing and random effects analyses of the functional data were performed with Brain Voyager QX software (Brain Innovation, Maastricht, The Netherlands). Before the analyses were completed the following preprocessing steps were performed. Three-dimensional motion correction to correct for small head movements was completed by alignment of all volumes to the first volume. Estimated translation and rotation movements never exceed 2 mm for any subject in these analyses. Further data preprocessing comprised of linear trend removal and filters for spatial (4 mm full-width half-maximum isotropic Gaussian kernel) as well as temporal (high pass filter: 1 cycle/run) smoothing to remove aliased signal correlated with background respiration and heart rate. Anatomical and functional images were co-registered and normalized to Talairach space. Statistical analysis was performed by multiple linear regression of the signal time course in each voxel. The expected BOLD signal change for each condition within a run was modeled by a canonical hemodynamic response function.
fMRI analyses involved deriving one mean image per individual for each relevant contrast in the activation task by subtracting the baseline scan from the post-treatment scan (e.g., Post-treatment – Baseline) after accounting for the hemodynamic response function. These contrast images were further analyzed to examine the effects of estradiol compared to placebo treatment using standard independent samples t-test procedures in Brain Voyager. To correct for multiple comparisons, we used the cluster-level statistical threshold estimator from Brain Voyager QX to estimate a minimum cluster size threshold based on the approach of Forman, Cohen, Fitzgerald, Eddy, Mintun, and Noll (2005). The starting p-value used in this procedure was p < 0.005. This procedure estimated a minimum cluster size of 6 at an alpha level of 0.01.