Variations in levels of estrogens are known to affect various behaviors in rodents. Thus, it is important to take into consideration stages of the estrous cycle when assessing cognitive function in female rodent subjects (Locklear & Kritzer, 2014). The majority of estrogens are cyclically produced by the ovaries and the estrus cycle occurs in four-phases: proestrus, estrus, metestrus, and diestrus. Levels of estrogens are low during metestrus, steadily rise during diestrus, peak during proestrus, and drop below baseline during estrus (Nilsson et al., 2015). Importantly, these naturally occurring short-term fluctuations in steroid hormones can cause changes in synaptic density. For example, females in proestrus show greater numbers of apical dendritic spines in hippocampal CA1 pyramidal neurons compared to females in the diestrus or estrus phases of the estrous cycle (Gonzalez-Burgos et al., 2004; Kinsley et al., 2005; Woolley et al., 1990). Exogenous administration of estrogen is also known to modulate several brain regions, such as the hippocampus and prefrontal cortex, that are important for learning and memory (Becker et al., 2002; Frick, 215; Frick et al., 2018), and estrogen-induced neuroplasticity in these brain regions has been empirically linked to cognitive function (Barha and Galea, 2010, Brinton, 2009, Woolley, 1998). Thus, one aim of the present study was to investigate how fluctuations of natural estrogens throughout an estrous cycle affect performance in the Open Field Tower Maze (OFTM), a hippocampus-dependent spatial navigation task intentionally designed to minimize stress (Lipatova et al., 2015, Lipatova et al., 2020).

Studies have shown that both exogenous administration of estrogen to ovariectomized rats (Daniel et al., 1997, Fader et al., 1998, Gibbs, 1999, Gibbs, 2000, Gibbs et al., 2004, Hammond et al., 2009; Lipatova et al., 2013, 2014; Locklear and Kritzer, 2014, Tuscher et al., 2015) and naturally cycling estrogens (Frye et al., 2007, Walf et al., 2009) have beneficial effects on hippocampus-dependent tasks. While there is an abundance of behavioral research supporting the beneficial effects of estrogen on cognitive function (see Luine & Frankfurt, 2020 for recent review), it is notable that some studies have previously found learning impairments associated with estradiol replacement and naturally cycling levels of estrogens (Berry et al., 1997, Diaz-Veliz et al., 1991, Dı́az-Véliz et al., 2000, Gresack and Frick, 2006, Snihur et al., 2008; Van Oyen et al., 1979; Warren & Juraska, 1997). Importantly, these studies used experimental paradigms that include stressful or aversive elements. The presence of a stressor is a potential factor that may help explain conflicting findings about the role of estrogens in cognitive processing. Stress has been shown to negatively impact neural processing within the hippocampus, which correlates with spatial learning deficits (Kim et al., 2007, Beck and Luine, 2010). Restraint stress specifically has been found to impair rodents’ spatial performance during both acquisition and retention (Sunanda & Raju, 2000). In fact, inconsistencies in sex-specific hippocampal function (Koss & Frick, 2017) may be partially explained by sex differences in stress sensitivity. In the Morris Water Maze (MWM), a spatial paradigm that is inherently stressful (Harrison et al., 2009), female rats travel longer distances and need more time to reach a hidden platform than male rats. These differences are due to females spending more time in the periphery of the maze, which is indicative of a greater stress response (Beiko et al., 2004). Similarly, female rats also spend more time than male rats in the perimeter of a Barnes Maze (Locklear & Kritzer, 2014), another maze that has been shown to increase corticosterone levels (Harrison et al., 2009). It is unclear whether this female-specific stress sensitivity during spatial learning is hormone-dependent. The present investigation aims to determine how elevated stress response during spatial learning affects female rats across the different stages of the estrous cycle.

Estrogens’ influence on a female’s stress response seems to depend on whether the stress exposure is long-term (chronic) or short-term (acute). There is evidence for decreased anxiety-like behavior in rodents during naturally high levels of cycling estrogens. For example, the rats spend a greater amount of time in the open arms of the elevated-plus maze during proestrus, compared to the rats in other phases of the estrous cycle, when levels of estrogens are lower (Walf et al., 2009, Frye et al., 2000, Marcondes et al., 2001). Moreover, research indicates that estrogen protects cognitive function against the impairing effect of long-term exposure to chronic stressors (Luine, 2016, Conrad et al., 2003). However, in the presence of acute, novel stress estrogens appear to induce adverse effects on cognitive performance. Shanksy et al. (2004) found that an increase in estrogens elevates female rats’ stress response, which hinders learning. Female rats in the proestrus phase, characterized by high levels of estrogens, required significantly lower dosages of an anxiogenic drug to experience working memory impairments relative to males, but not during estrus, when the levels of estrogens are lower (Shansky et al, 2004). Thus, it is possible that while estrogens reduce general anxiety-like behavior in female rats, they increase fear-like behavior in response to an acute stressor, which leads to cognitive impairment. The present investigation specifically tested the effects of an acute stressor on hippocampus-dependent learning in female rats during high and low naturally cycling levels of estrogens.

The open-field tower maze (OFTM) is used to examine acquisition and retention of spatial learning in the present study. The OFTM was designed to measure hippocampus-dependent spatial navigation similarly to the Morris Water Maze and Barns Maze, while intentionally minimizing stressors such as water immersion or exposure to bright lights. Cycling exogenous estrogen treatment has been previously shown to enhance the rate of place learning and retention in the OFTM (Lipatova, et al., 2013). Additionally, there are no sex differences in acquisition of spatial learning in the OFTM, but acute stress differentially impacts the expression of retained spatial memory in female compared to male rats (Lipatova et al., 2018). More recently, we demonstrated that place learning in the OFTM is hippocampus-dependent (Lipatova et al., 2020). Thus, the OFTM is a well-suited paradigm to study the impact of acute stress on hippocampus-dependent learning when rats are at different stages of the estrous cycle. The percent of first-choice correct response was used as a non-locomotor-dependent and non-anxiety-dependent indicator of successful learning and retention on the OFTM (Lipatova et al., 2015).

In addition to measuring hippocampus-dependent spatial ability, the current study analyzed other important behaviors that may be influenced by the cycling levels of estrogens and response to stress. The modulation of these behaviors may contribute to the mediation of the performance in the spatial navigation task. For example, duration of time spent in the outer perimeter of the arena (i.e. “wall hugging”), immobility state (i.e. “freezing”) and increased latency to first center region entry are all documented indicators of stress-like behaviors in rodents (Treit & Fundytus, 1988, Pham et al., 2009, Belviranli et al., 2012). Velocity, distance moved, and acceleration are all useful measures of analyzing locomotor activity (Noldus et al., 2001). Thus, in the present investigation we tracked these explorative behaviors of the cycling females rats as they were trained in the OFTM.

In summary, evidence suggests that interactions between cycling female hormones and acute stress influence cognitive function and exploratory behaviors. The present investigation used the OFTM to assess hippocampus-dependent navigation in female rats. Analysis of vaginal cells was used to track the different estrous phases throughout all training and testing. Acute stress was induced using physical restraint along with exposure to bright lights. Importantly, all acquisition trials were given within a single session to ensure that the stress exposure was novel and acute, and to limit the training to a single phase of the estrous cycle.