COMPARISON OF THE EFFECTS OF ESTRADIOL AND PROGESTERONE ON SEROTONERGIC FUNCTION

INTRODUCTION

Depression is more common in females than males (1) and in women it may be more common at times of falling estradiol levels (2). Women tend to report more depressive and dysphoric symptoms, even if not full-blown major depressive disorder (MDD), during times of large hormonal changes, including menopause, the premenstrual period and postpartum which suggest that hormonal changes are playing a role in these symptoms (3, 4). Nevertheless, the effectiveness on depressed mood of hormone replacement therapy during the postmenopausal period is somewhat controversial and both positive (5) and negative (6) results have been reported.

In ovariectomized rats, we have shown that acute systemic administration of either estradiol benzoate (EB) and/or progesterone (P) blocked the ability of SSRIs, fluvoxamine and citalopram, to inhibit the function of what is widely considered their initial cellular target in brain – the serotonin transporter (SERT) (7). In addition, when given systemically, EB, but not P, blocked the function of the SERT (8).

The behavioral consequences of these hormonal effects are not known. The most widely-used behavioral test in rats that is predictive of antidepressant efficacy in patients is the forced swimming test (FST) (9, 10). AD-like effects of estradiol and progesterone have been shown in the FST (11-13). However, few studies have examined if ovarian steroids alter the effects of ADs in these tests. In the first part of this study, the effects of acute ovarian hormones on the ability of fluvoxamine to decrease immobility in the FST were examined.

A major advance in understanding estrogen’s actions was the discovery and cloning in 1996 of a second estrogen receptor (ER), ERβ (14), that is genetically distinct from the ERα subtype first cloned ten years earlier (15). Recently, a novel membrane-associated ER, GPR30, was identified (16, 17). GPR30 is a seven transmembrane-spanning G-protein coupled receptor that promotes rapid estrogen signaling in a variety of cell types (18). GPR30 shows specific high affinity binding to estradiol and related estrogens (19). A second part of this study examined the mechanisms underlying the effects of ovarian hormones on SERT function and on the ability of fluvoxamine to block SERT function, using in vivo chronoamperometry after local application of hormones, hormone receptor-subtype specific agonists and antagonists. The use of local application provides more specificity for mechanistic studies as both hormone and drug effects are primarily confined to the area into which they are administered. All in vivo chronoamperometry experiments were carried out in the CA₃ region of the hippocampus as under our experimental conditions, the active clearance of 5-HT in this area is mediated primarily by the SERT (20). Further, there is evidence for the involvement of the hippocampus in depression and the effects of antidepressants (21). Finally, both intracellular and membrane ERs in the hippocampus can mediate hippocampal effects of estrogen (22).

METHODS AND MATERIALS

Ovariectomized (OVX) rats (Sprague-Dawley; 250-350g, Harlan, Houston, TX) were housed on a 12:12h light/dark cycle with lights on at 0700. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Institutional Animal Care and Use Committee. OVX rats were used 2-3 weeks after surgery.

RESULTS

DISCUSSION

These results demonstrate that behaviorally, in OVX rats treated with either EB or P or their combination, fluvoxamine has a markedly reduced ability to induce an antidepressant-like effect in the FST, consistent with these hormones blocking the ability of fluvoxamine to slow 5-HT clearance after similar treatment. Local application of E₂ decreases SERT function as the clearance of exogenously applied 5-HT is slower and also inhibits fluvoxamine’s effect on 5-HT clearance, at both early and later time points. Studies with an ER antagonist indicate that this effect of E₂ on 5-HT clearance is distinct from its ability to block the inhibitory effect of fluvoxamine on 5-HT clearance. Indeed, studies with ER subtype-selective agonists show that these two effects of E₂ are mediated by different ER subtypes. ERβ and GPR30 may play a role in the effects of E₂ on basal clearance whereas its blockade of fluvoxamine’s effect on 5-HT clearance is mediated through activation of ERα. Progesterone has a different effect on SERT function as its local application had no effect on 5-HT clearance but it does block the ability of fluvoxamine to slow 5-HT clearance, only at the later time point, and this effect is prevented by blockade of the progesterone receptor.

Differences have been seen between males and females in the FST. Females, at all stages of the estrous cycle, are reported to be less immobile than males in the FST (36), or to be less immobile only during proestrus (37). AD-like effects of estradiol in the FST, similar to those produced by SSRIs, were demonstrated in rats and in mice (11,12). By contrast, the AD-like effect of progesterone was similar to that of selective noradrenergic reuptake inhibitors (i.e. decreased immobility and increased climbing behavior) (13). In the present study, neither estradiol nor progesterone alone or combined, administered once to OVX rats, had an AD-like effect in the FST (Figure 2). It is likely that this difference with the findings cited above could be due to variability in the treatment paradigms used, such as the time elapsed between treatment and the swim test and the duration of hormonal treatment.

Ovarian hormones can act either by a genomic mechanism, using classical nuclear transcription factor receptors requiring at least 30-60 minutes to be manifest and is associated with changes in protein synthesis (38), or by a non-genomic, less well-characterized, rapid (seconds to minutes) membrane receptor signaling mechanism (39-42). Steroid- bovine serum albumin (BSA) conjugates are useful tools to study the membrane-mediated action of steroids since they are biologically active and do not penetrate readily the plasma membrane of the cell (43,44). We used receptor agonists and antagonists, hormone-BSA conjugates and the time course of hormone responses (earlier vs later) to try to elucidate those hormone receptors responsible for the effects observed.

In addition to interfering with the effect of fluvoxamine, estrogen given either systematically (8) or locally had an inhibitory effect of its own on 5-HT clearance (Figures 3 and 4). Although fluvoxamine no longer slowed 5-HT clearance in OVX rats administered estradiol systematically or locally, the inhibitory effect of estradiol alone on 5-HT clearance may have produced a “ceiling effect” so as to compromise any further inhibitory effect of fluvoxamine. However, some of the data presented in Figure 5 argue against this possibility. Most importantly, at 10 min the receptor antagonist, ICI 182,780, restored the inhibitory effect of fluvoxamine even though basal clearance still remained inhibited. This makes it unlikely that the ability of E₂ to inhibit the effects of fluvoxamine was due to a “ceiling effect” and indicates that the inhibitory effect of E₂ alone on 5-HT clearance is not due precisely to the same mechanism by which it inhibits the effect of fluvoxamine. This was further confirmed by the use of ER subtype-specific agonists (see below). From the time course of E₂’s effects, together with those of the E₂-BSA conjugate reported previously (8), as well as the effects of ICI 182,780, it may be inferred that E₂ has (1) rapid, presumably non-genomic effects, only one of which is mediated by a receptor antagonized by ICI 182,780; and (2) possibly later genomic effects, both of which can be blocked by ICI 182,780. It is not known which ER subtype is responsible for mediating these effects.

E₂ has actions at both types of classical nuclear receptors, ERα and ERβ. Some of the regional differences in ERα versus ERβ distribution in brain may confer specific functional effects of E₂ acting via these receptor isoforms. ERα is predominant in the ventromedial hypothalamus and is related to E₂’s facilitatory actions on female rodent sexual responding; ERβ is predominant in limbic and/or stress-responsive regions of the brain, e.g., hippocampus, bed nucleus of the stria terminalis, and paraventricular nucleus of the hypothalamus (45). Thus, E₂ may act to alter affective behaviors of rodents in part through activation of ERβ. Effects of E₂ in producing anxiety- and/or depression–like behaviors through activation of the ERβ subtype have been reported using both ERβ knockdown and ERβ activation approaches (12, 46-49). It has also been shown that the AD-like effect of estradiol was mediated by activation of ERβ, rather than ERα (12, 50). Consistent with this, our data indicate that the AD-like effect of E₂ (i.e., slower 5-HT clearance) was due to ERβ activation as DPN, its specific agonist, mimicked this effect (figure 6). By contrast, E₂’s blockade of fluvoxamine’s ability to slow serotonin clearance was mimicked only by ERα agonists, PPT and THC, and seems to be mediated through this receptor subtype (Figure 7). Although it is established that the AD-like effect of E₂ is mediated via ERβ, to our knowledge this is the first report showing that E₂’s activation of ERα interferes with the ability of an SSRI to block SERT function.

E₂ acts not only at classic nuclear ERs but also at the novel G protein-coupled ER, GPR30. In hypothalamus, GPR30 was detected in the paraventricular nucleus and supraoptic nucleus (51). The expression of GPR30 in rats was observed at both mRNA and protein levels in the hippocampus (52). Recently, GPR30 stimulation was shown to attenuate serotonin receptor signaling in the PVN as demonstrated by reduced oxytocin and ACTH responses, suggesting that GPR30 may play a role in mood disorders (53). The activation of GPR30 by its specific agonist, G1, mimicked the effects of E₂ in slowing 5-HT clearance whereas it had no effect on the fluvoxamine-induced blockade of 5-HT clearance (Figures 6 and 7). Thus, the effect of E₂ on slowing basal 5-HT clearance involves either ERβ and/or GPR30. With regards to signaling, although the nuclear receptors are known to regulate gene transcription they are also capable of activating second messenger signaling pathways such as mitogen activated protein kinases (MAPK), calcium/calmodulin-dependant protein kinases (CamKII) and cAMP response element-binding proteins (CREB) (54, 55). Future studies will be necessary to clarify which signaling pathway(s) are involved in the observed effects of E₂ on SERT function. GPR30 functions as a classic G protein coupled receptor. Studies using breast cancer cells demonstrated E₂ signaling through GPR30-mediated activation of MAPK through transactivation of epidermal growth factor (EGF) receptor and stimulation of adenyl cyclase and ERK phosphorylation (56, 57). Whether GPR30 utilizes the same signaling processes in the brain remains to be evaluated.

Progesterone and its receptors play a pivotal role in the regulation of brain activity by its modulation of synthesis and release of neurotransmitters and neuropeptides in response to physiological or pathological stimuli (58, 59). Progesterone altered SERT function in a somewhat different manner than estradiol did. Local application of progesterone mimicked the effect of its systemic administration in that it had no effect on 5-HT clearance (Table 1) whereas it blocked the ability of fluvoxamine to slow 5-HT clearance at later time points (Figure 8). This inhibitory effect of progesterone was blocked by its receptor antagonist, RU486. Further the P-BSA conjugate had no effect on the ability of fluvoxamine to slow clearance (Figure 8). Taken together, these results indicate that the effect of P on fluvoxamine’s ability to slow clearance does not involve membrane PRs but are mediated via activation of intracellular PRs. Progesterone can also influence neuronal activity through its metabolite allopregnanolone, a neurosteroid which acts as an agonist on the GABA_A receptor (60-62). However, a direct effect of P on its receptors is suggested by the ability of RU 486 to block the inhibitory effect of P and by the fact that PRs are demonstrated to be expressed in the hippocampus (63-65).

Although acute treatment with estrogen decreased 5-HT clearance in the CA₃ region of the hippocampus, it did not alter total (both membrane-bound and intracellular) SERT density as measured by the binding of [³H]cyanoimipramine (8). However, the possibility of changes in membrane distribution of SERT cannot be ruled out. Estrogen-regulated changes in SERT expression have been reported in female macaques and rats brain (66, 67). Beside acting on the transporter, estrogen can act at several levels of the serotonergic system, including effects on its synthesis, receptors and function; this could account for estrogen having beneficial effects on mood that are mediated, at least in part, by interaction with serotonergic systems (68-74).

Estrogen enhancement of plasticity is evidenced by increases in neurogenesis, neural network connectivity and synaptic transmission (75). Hormonal (or estrogen alone) therapy has also been reported to have antidepressant effects in perimenopausal and postmenopausal women (5), although this is not a universal finding (6). Of specific importance for this study is the effect of addition of ovarian hormones, particularly estrogen, on the efficacy of SSRIs in patients. The results of such studies are quite variable. There is certainly no consistent body of data demonstrating that elderly depressed women taking estrogen have a better antidepressant response to SSRIs than those not taking the hormone (76-80). Our experiments do not mimic the clinical situation as we are studying the acute interaction of the ovarian hormones with SSRIs. However, the results obtained in our studies in OVX rats show that, when given acutely, estrogen (or progesterone) inhibited the ability of SSRIs to block the serotonin transporter, which could result in a diminution of SSRI efficacy. This effect of estradiol was mediated through ERα activation. By contrast, estrogen’s blockade of 5-HT clearance, which may result in some beneficial enhancement of serotonin function, appears to be a consequence of ERβ and/or GPR30 stimulation. Thus, understanding the mechanisms behind such effects might reveal a strategy, such as targeting of ERβ or GPR30, to permit beneficial effects of estrogen without its deleterious effect on SSRI-induced efficacy.