These experiments were designed to adapt a previously described PWD procedure for behavioral analysis, and to determine whether the behavioral effect of PWD could be replicated by withdrawing 5α-reduced neurosteroids (such as ALLO). Experiment 1a determined that three days of PWD was required to detect a significant increase in FST immobility. This finding is consistent with recent reports of delayed increases in FST immobility following steroid withdrawal (Bekku et al., 2007
; Stoffel & Craft, 2004
), and of increased depression-like behavior in rats at three days postpartum, as assessed in the differential reinforcement of low response-rate model of depression (Molina-Hernández et al., 2000
). In the case of Stoffel and Craft (2004)
, rats received progesterone and estradiol injections, separately or in combination, for a period of more than three weeks. Bekku and colleagues (2007)
used multiple strains of mice (ICR, C57BL/6J, DBA/2N, and CD-1) and assessed the effect of ovariectomy on FST immobility. Although their results varied from the current data in some ways, such differences may be the result of differences in methodology. Importantly, despite differences in methods, each of these studies reported an effect of steroid withdrawal on FST that is consistent with the current work.
Experiment 1b was used to confirm the effects of this procedure on plasma progesterone levels. Plasma progesterone concentrations were significantly increased at 0.5 hr after injection and remained elevated at 2 hr after injection when compared to levels observed in mice treated with vehicle or withdrawn for three days. Progesterone levels two hours after a single progesterone injection (2 hr group) or two hours after repeated progesterone injections (0 d PWD group) did not differ, suggesting similar kinetics of progesterone during this procedure. This experiment also demonstrated a significant decrease in plasma progesterone levels at three days of withdrawal from repeated progesterone injections, but not at one day of withdrawal. However, plasma progesterone levels were not different between vehicle-treated mice and mice allowed to withdraw from progesterone for three days.
These findings indicate that there is not a simple relationship between progesterone levels and FST immobility. Comparing the results of Experiments 1a and 1b, these data show that low levels of progesterone were not associated with increased FST immobility among vehicle-treated mice, but that similarly low levels of progesterone were associated with significantly increased FST immobility following withdrawal from high levels of progesterone. Taken in conjunction with the finding that an abrupt drop in progesterone levels was associated with increased FST immobility (Saavedra et al., 2006
, see Introduction for experimental methods), these data suggest that a withdrawal-induced change in progesterone levels reflects neuroadaptation to high levels of progesterone or its metabolites.
Despite the temporal continuity between hormonal fluctuations and PMS symptoms, several reports have suggested that hormones are normal throughout the cycle in women with PMS (Halbreich, 2003
). Thus, it is of great interest to explore hormone changes outside the estrogen/progesterone dichotomy. One potential mechanism through which PWD might result in depression is through a corresponding decrease in its GABAergic metabolite ALLO. This hypothesis was tested by administering the 5α-reductase enzyme inhibitor finasteride, which produces a concomitant decrease in 5α-dihydroprogesterone and ALLO. In Experiment 2, mice that received 100 mg/kg finasteride and progesterone exhibited a significant increase in FST immobility versus mice that received continuous progesterone. Also, the degree to which FST immobility was increased by 100 mg/kg finasteride administration was consistent with the degree of FST immobility produced by three days of PWD. Since progesterone levels should not be decreased in the mice receiving progesterone and finasteride injections, these results demonstrate that withdrawal of 5α-reduced steroids can mimic the behavioral effects of PWD. Thus, these findings support the hypothesis that the effects of PWD on FST immobility are mediated through ALLO withdrawal.
Several lines of evidence indicate that the progesterone injection regimen utilized in the present study produces fluctuations in ALLO levels that are temporally related to progesterone levels, and that the use of finasteride can block the progesterone-induced increase in ALLO levels. First, in intact female mice and rats 3–5 daily progesterone injections produced high-physiological levels of ALLO (Costa et al., 1995
; Gulinello & Smith, 2003
) that are significantly decreased during PWD. Second, data from our laboratory has shown an 80% decrease in brain ALLO levels at 24 hr following injection of the 50 mg/kg dose of finasteride in male mice (for a review on finasteride see Finn et al., 2006a
), which is consistent with data in rats (VanDoren et al., 2000
). Since female mice have higher endogenous ALLO levels than male mice (Finn et al., 2004b
) and since we administered exogenous progesterone, we presumed that the 100 mg/kg dose of finasteride would be more efficacious at decreasing endogenous ALLO levels. Third, the 50 mg/kg dose of finasteride significantly decreased ALLO levels following a single progesterone injection (Rhodes and Frye, 2005
), whereas the 100 mg/kg finasteride dose significantly reduced ALLO levels by 85% in pseudopregnant female rats without altering plasma progesterone concentrations (Reddy et al., 2001
). Collectively, these findings suggest that the use of the 100 mg/kg dose of finasteride in the present study should significantly reduce ALLO to levels that have been reported to occur during PWD.
The present work adds to a line of research indicating a role for ALLO in depression-like behavior in mice. For example, Frye and coworkers (2004)
previously reported that progesterone decreased FST immobility in wildtype mice but not in mice with a null mutation for the Srd5a1
gene (encodes the type 1 5α-reductase enzyme). Also, systemic or intra-hippocampal administration of finasteride (Frye & Walf, 2002
) as well as intra-amygdala injections of finasteride (Walf et al., 2006
) increased FST immobility in female rats. These bidirectional manipulations of ALLO levels provide evidence for an inverse relationship between ALLO levels and FST immobility, albeit not in the context of PWD. In contrast, the efforts of other researchers (e.g., Bekku et al., 2007
; Molina-Hernández et al., 2000
) have reported depression-like effects of PWD, but without specifically assessing potential mechanisms underlying the effect. Thus, FST immobility was previously studied in the context of acute ALLO manipulation or progesterone withdrawal, and the current research combines these lines of research.
Anxiety-like behaviors have been better characterized within the context of PWD. Interestingly, while onset of FST immobility appears to be delayed by some number of days, rodent models of anxiety such as the elevated plus maze (EPM) show anxiogenic-like responses as early as one day following PWD. For example, Bitran and Smith (2005)
induced PWD by ovariectomizing rats after 10 days of hormonally-induced pseudopregnancy and found that one day of PWD increased anxiety-like behavior in the EPM compared to ovariectomized control rats. Similar effects in the EPM were detected in rats after one day of PWD when progesterone was administered via implantation of a progesterone-filled, silicone capsule, and withdrawal was achieved by removing the capsule (Smith et al. 2004
). Finally, three injections of progesterone (5 mg/kg) or ALLO (10 mg/kg) significantly increased anxiety-like behavior in the EPM at 3–4 hrs after the last injection in male and female mice (Gulinello & Smith, 2003
). Differences between the effects of ALLO and progesterone on anxiety and depression may have relevance to syndromes such as PMS, which has been characterized as having highly variable symptom profiles (Chrisler & Levy, 1990
; Gotts et al., 1995
; Halbreich, 2003
It should be noted that research examining animal models of PWD has utilized several methods to manipulate progesterone levels (e.g., injections, osmotic capsules, pseudopregnancy, ovariectomy). In addition, different species of rodents and different strains within a given species can complicate direct comparisons among these studies. However, despite variation in progesterone methods, choice of animals, and other methodological details, many groups have found consistent effects of steroid withdrawal on rodent behavior in models of anxiety or depression. Thus, the effects of PWD appear to be robust, despite methodological diversity.
These studies demonstrate that the present model of PWD, chosen to reflect the shorter period of exposure to progesterone during the menstrual cycle (as opposed to pregnancy), is effective at increasing depression-like behavior in female mice from an inbred strain. The procedures are quick and easy to perform, requiring only eight days of experimentation, and produce behavioral differences without resorting to surgeries or manipulation of non-progestin steroids such as estrogens. While not definitive, the results of Experiments 2 offer support for the hypothesis that ALLO withdrawal may contribute to depression-like behavior associated with PWD. This model has the potential to be incorporated into a variety of experimental procedures that may further elucidate electrophysiological changes in the nervous system, neuroanatomical correlates of behavioral changes, and pharmacological dissociations of the substrates underlying depression-like behavior following PWD.