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. 2022 May 25:16:885321.
doi: 10.3389/fnins.2022.885321. eCollection 2022.

Evaluating the Cognitive Impacts of Drospirenone, a Spironolactone-Derived Progestin, Independently and in Combination With Ethinyl Estradiol in Ovariectomized Adult Rats

Stephanie V Koebele  1   2 , Mallori L Poisson  1   2 , Justin M Palmer  1   2 , Claire Berns-Leone  1   2 , Steven N Northup-Smith  1   2 , Veronica L Peña  1   2 , Isabel M Strouse  1   2 , Haidyn L Bulen  1   2 , Shruti Patel  1   2 , Corissa Croft  1   2 , Heather A Bimonte-Nelson  1   2
Affiliations

Evaluating the Cognitive Impacts of Drospirenone, a Spironolactone-Derived Progestin, Independently and in Combination With Ethinyl Estradiol in Ovariectomized Adult Rats

Stephanie V Koebele et al. Front Neurosci. .

Abstract

Oral contraceptives and hormone therapies require a progestogen component to prevent ovulation, curtail uterine hyperplasia, and reduce gynecological cancer risk. Diverse classes of synthetic progestogens, called progestins, are used as natural progesterone alternatives due to progesterone's low oral bioavailability. Progesterone and several synthetic analogs can negatively impact cognition and reverse some neuroprotective estrogen effects. Here, we investigate drospirenone, a spironolactone-derived progestin, which has unique pharmacological properties compared to other clinically-available progestins and natural progesterone, for its impact on spatial memory, anxiety-like behavior, and brain regions crucial to these cognitive tasks. Experiment 1 assessed three drospirenone doses in young adult, ovariectomized rats, and found that a moderate drospirenone dose benefited spatial memory. Experiment 2 investigated this moderate drospirenone dose with and without concomitant ethinyl estradiol (EE) treatment, the most common synthetic estrogen in oral contraceptives. Results demonstrate that the addition of EE to drospirenone administration reversed the beneficial working memory effects of drospirenone. The hippocampus, entorhinal cortex, and perirhinal cortex were then probed for proteins known to elicit estrogen- and progestin- mediated effects on learning and memory, including glutamate decarboxylase (GAD)65, GAD67, and insulin-like growth factor receptor protein expression, using western blot. EE increased GAD expression in the perirhinal cortex. Taken together, results underscore the necessity to consider the distinct cognitive and neural impacts of clinically-available synthetic estrogen and progesterone analogs, and why they produce unique cognitive profiles when administered together compared to those observed when each hormone is administered separately.

Keywords: cognition; contraceptive; drospirenone (DRSP); ethinyl estradiol (EE); rat model.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Experiment 1 Water radial-arm maze. (A) During Early Acquisition, all groups treated with drospirenone had improved WMC performance compared to rats without hormone treatment when working memory load was moderately taxed. (B) This working memory benefit was extended to WMI errors on the moderate working memory load trial for DRSP-Low and DRSP-Medium groups compared to rats without hormone treatment. (C) During the Asymptotic Phase, DRSP-Medium continued to enhance WMI performance compared to rats without hormone treatment on Trial 4, the maximum working memory load trial. Rats treated with Vehicle (D) or DRSP-Low (E) showed impaired delayed memory retention on Trial 3 following a 6-h delay compared to the previous day’s baseline performance. Rats treated with (F) DRSP-Medium and (G) DRSP-High treatment did not exhibit a statistically significant delay-related impairment. *p < 0.05 and **p < 0.01. Vehicle n = 10, DRSP-Low n = 10, DRSP-Medium n = 10, and DRSP-High n = 10.
FIGURE 2
FIGURE 2
Experiment 1 Morris water maze. Across all days of testing (A), rats treated with the DRSP-Medium dose (C) and the DRSP-High dose (D) swam less distance to reach the platform compared to rats without hormone treatment, while the DRSP-Low group (B) performed similarly to the Vehicle group. (E) On the probe trial, all groups localized to the previously platformed target quadrant. *p < 0.05 and ****p < 0.0001. Vehicle n = 10, DRSP-Low n = 10, DRSP-Medium n = 10, and DRSP-High n = 10.
FIGURE 3
FIGURE 3
Experiment 1 Western blot protein analysis. (A) Representative images of IGF1-R, GAD65, and GAD67 western blot analysis within the dorsal hippocampus, entorhinal cortex, and perirhinal cortex. There were no Treatment differences in IGF1-R expression (B), GAD67 expression (C), or GAD65 expression (D) for any brain region evaluated. Vehicle n = 10, DRSP-Low n = 10, DRSP-Medium n = 10, and DRSP-High n = 10.
FIGURE 4
FIGURE 4
Experiment 2 Water radial-arm maze. (A) During early acquisition, rats treated with drospirenone only showed enhanced WMI performance on the moderate working memory load trial compared to rats without hormone treatment, or rats given a combination of drospirenone plus a low or high dose of EE. (B) During the Late Acquisition Phase, rats treated with a combination of drospirenone and high EE had enhanced WMI performance on the moderate working memory load trial compared to rats treated with a high dose of EE only. (C) Rats treated with a combination of drospirenone and low EE showed impaired performance on the maximum working memory load trial compared to a low dose of EE alone during Late Acquisition. (D) Rats without hormone treatment did not exhibit a delay-induced memory impairment, (E,F,H) while rats treated with EE-Low, EE-High, and DRSP + EE-Low were significantly impaired following a 6-h delay. (G,I) The DRSP group and the DRSP + EE-High group were not significantly impaired following a 6-h delay. *p < 0.05 and **p < 0.01. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
FIGURE 5
FIGURE 5
Experiment 2 Morris water maze. (A) There were no Treatment differences across MM Days 1–5. (B) All groups localized to the target quadrant during the probe trial. ****p < 0.0001. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
FIGURE 6
FIGURE 6
Experiment 2 Open field task performance. Performance varied across: (A) Total Distance Moved, (B) Center Distance, (C) Small Center Distance, (D) Corner Distance, (E) Center Time, (F) Small Center Time, and (G) Corner Time, in the OFT. In general, the EE-High group exhibited decreased anxiety-like behavior in the Open Field. *p < 0.05 and **p < 0.01. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
FIGURE 7
FIGURE 7
Experiment 2 Body weight and uterine weight. All subjects were ovariectomized. (A) Rats treated with EE alone and in combination with drospirenone weighed significantly less than rats without hormone treatment. Rats treated with drospirenone weighed more than those that received drospirenone plus EE at a low or high dose. (B) Uterine weights from rats treated with EE weighed significantly more at euthanasia compared to rats without hormone treatment. Rats treated with drospirenone had lower uterine weights compared to rats treated with a combination of drospirenone and EE, but did not differ from rats without hormone treatment. ****p < 0.0001. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
FIGURE 8
FIGURE 8
Experiment 2 Western blot protein analysis. (A) Representative images of IGF1-R, GAD65, and GAD67 western blot analysis within the dorsal hippocampus, entorhinal cortex, and perirhinal cortex. (B) There were no differences in IGF1-R expression between groups in any brain region evaluated. (C) Rats treated with a low dose of EE or drospirenone plus a high dose of EE showed increased GAD67 expression in the perirhinal cortex compared to rats without hormone treatment. No differences in GAD67 were detected between groups in the dorsal hippocampus or entorhinal cortex. (D) Rats treated with a low dose of EE had increased GAD65 expression in the perirhinal cortex compared to rats without hormone treatment. No differences in GAD65 were detected between groups in the dorsal hippocampus or entorhinal cortex. *p < 0.05 and **p < 0.01. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
FIGURE 9
FIGURE 9
Experiment 2 Correlations between proteins of interest and WRAM performance. (A) Within the EE-High group, greater GAD65 expression in the entorhinal cortex was associated with fewer WMC errors when working memory was moderately taxed during the Early Acquisition Phase of WRAM. (B) Within the DRSP + EE-Low group, greater IGF1-R expression in the perirhinal cortex was associated with more WMC errors when working memory was moderately taxed during the Early Acquisition Phase of WRAM. (C) Within the DRSP + EE-High group, greater GAD67 expression in the perirhinal cortex was associated with increased WMC errors on the maximum working memory load trial during Early Acquisition. (D) Within the DRSP + EE-High group, greater GAD65 expression in the perirhinal cortex was associated with decreased WMI errors on the moderate working memory load trial during Early Acquisition. Vehicle n = 10, DRSP n = 10, EE-Low n = 10, EE-High n = 10, DRSP + EE-Low n = 10, and DRSP + EE-High n = 10.
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