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. 2016 Dec 8:10:517.
doi: 10.3389/fnins.2016.00517. eCollection 2016.

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions

Ryoko Hiroi  1 , Giulia Weyrich  1 , Stephanie V Koebele  1 , Sarah E Mennenga  1 , Joshua S Talboom  1 , Lauren T Hewitt  1 , Courtney N Lavery  1 , Perla Mendoza  1 , Ambra Jordan  1 , Heather A Bimonte-Nelson  1
Affiliations

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions

Ryoko Hiroi et al. Front Neurosci. .

Abstract

Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17β-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of behavior. These distinct associations imply that cognition, anxiety-like, and depressive-like behaviors are modulated by unique serotonergic neurocircuitry, opening the possibility of novel avenues of targeted treatment for different types of cognitive and affective disorders.

Keywords: Premarin; TpH2; cognition; estrogen; learning and memory; mood; serotonin; spatial.

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Figures

Figure 1
Figure 1
Time course of surgeries, hormone administration, behavioral testing, and sacrifice in the present study. Twenty-five days following Ovx, rats were randomly assigned to one of the following treatment groups: Vehicle (sesame oil, n = 9), CEE (30 μg CEE in sesame oil, n = 8), or E2 (3 μg E2 in sesame oil, n = 8). One subcutaneous injection (0.1 mL volume) was given for 2 consecutive days, followed by 2 days off, and this pattern was repeated throughout the study until animals were sacrificed. Sixteen days following the initiation of hormone treatment (41 days after Ovx), a series of behavioral tests commenced in the following order: water radial arm maze (WRAM), Morris maze (MM), delayed-matched-to-sample (DMS), open field test (OFT), elevated plus maze (EPM), and forced swim test (FST). One week of rest prior to sacrifice was given to all animals following the last behavioral testing day to minimize any sub-acute stress effects on gene expression. Animals were sacrificed by decapitation and brains were rapidly removed, flash frozen in 2-methylbutane at −30°C, and stored at −80°C until processed for TpH2 mRNA analysis.
Figure 2
Figure 2
The effects of CEE and E2 on wet uterine weights (g) at sacrifice. To confirm Ovx and hormone treatment, uterine tissues were collected at sacrifice. The uterus was cut between the cervix and the ligature at the tips of the uterine horns remaining from Ovx, and trimmed of visible fat. The uterus was immediately weighed to obtain wet weight. Both CEE and E2 stimulated the uterus, as expected. *p < 0.0001.
Figure 3
Figure 3
The effects of CEE and E2 on the water radial arm maze (WRAM). Errors committed during the Acquisition Phase (days 2–7) for (A) working memory correct (WMC), (B) working memory incorrect (WMI), and (C) reference memory (RM). Errors committed during the Asymptotic Phase (days 8–12) for (D) WMC, (E) WMI, and (F) RM. *p < 0.05, #p < 0.10.
Figure 4
Figure 4
The effects of CEE and E2 on the water radial arm maze (WRAM) delay task. Working memory correct (WMC) errors committed by the vehicle (A), CEE (B), or E2 (C) groups during the post-delay trial (T3) following a 4-h delay between trials 2 and 3 on day 13. *p < 0.01.
Figure 5
Figure 5
The effects of CEE and E2 on the Morris maze (MM) and delayed-match-to-sample (DMS) behaviors. (A) Swim distance (cm) in the MM for days 1–5. (B) Percent swim distance in the target vs. opposite quadrants in the MM for the probe trial assessment. Total errors committed on DMS during (C) the Acquisition Phase (days 1–3) and (D) the Asymptotic Phase (days 4–7). *p < 0.05.
Figure 6
Figure 6
The effects of CEE and E2 on anxiety-like and depressive-like behaviors. Time spent in the center (A) and corners (B) and total distance traveled (C) in the open field test (OFT). Time spent in the open arms (D) and the closed arms (E) of the elevated plus maze (EPM). Time spent immobile (F) in the forced swim test (FST). *p < 0.05, #p < 0.10.
Figure 7
Figure 7
Subregional anatomy of the raphe nuclei. Top panels show the schematic diagrams (Paxinos and Watson, 1986) and middle panels show the representative photomicrographs of the subregions analyzed for TpH2 in situ hybridization signal in rostral, mid, and caudal levels of the midbrain raphe from each treatment group. Ovals and a triangle are shown on the bottom panels to illustrate the subregions analyzed for the densitometry. DRN, dorsal raphe nucleus; DM, dorsomedial; VM, ventromedial; DL, dorsolateral.
Figure 8
Figure 8
The effects of CEE and E2 on TpH2 mRNA in the subregions of the DRN. TpH2 mRNA in situ hybridization signal in the rostral (A) dorsomedial (rDM) and (B) ventromedial (rVM); medial (C) dorsomedial (mDM), (D) ventromedial (mVM), and (E) dorsolateral (mDL); caudal (F) dorsomedial (cDM), and (G) ventromedial (cVM) DRN. Each bar represents the mean ± SEM TpH2 optical density. *p < 0.05.
Figure 9
Figure 9
Correlation between TpH2 mRNA in the subregions of DRN and cognitive, anxiety-like, and depressive-like behaviors. Significant correlations using the Fisher's r- to z-test between TpH2 mRNA in the (A) rDM and total errors in the delayed-match-to-sample (DMS) task, (B) rDM and open arm time in the elevated plus maze (EPM), (C) rVM and immobility in the forced swim test (FST), (D) mDL and center time in the open field test (OFT), (E) mDM and open arm time in the EPM, (F) mVM and center time in the OFT, (G) cVM and open arm time in the EPM, and (H) cVM and immobility in the FST.
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