The Effects of Licorice Root Components on Cognition

THE EFFECTS OF LICORICE ROOT COMPONENTS ON COGNITION

PAYEL KUNDU

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Urbana-Champaign, 2018

Urbana, Illinois

Doctoral Committee:

Professor Susan L. Schantz, Chair Professor Janice M. Juraska Professor Jodi A. Flaws Associate Professor Joshua M. Gulley Associate Professor Lori T. Raetzman

ABSTRACT

A large body of research has shown that estrogens can modulate learning and memory.

Importantly, the effects of estrogens on cognition vary based on several factors including dose, the type of estrogen used, the age of the animal, the length of exposure, the route of exposure, as well as the demands of the cognitive task. Botanical estrogens are non-steroidal plant compounds that can mimic estrogens in the body by binding to estrogen receptors and affecting transcriptional changes. Many botanical estrogens are widely available over the counter in dietary supplements, despite a dearth of research about their effects. This work investigated the efficacy of components of licorice root, one of the most common sources of botanical estrogens in dietary supplements, to alter performance on three different cognitive tasks: a prefrontal cortex-sensitive delayed spatial alternation (DSA) task, a hippocampus-sensitive metric change in object location (MCOL) task and a striatum-sensitive double object recognition (DOR) task. Isoliquiritigenin (ISL), licorice root extract (LRE), and whole licorice root powder (LRP) were assessed. ISL is a pure compound found in licorice root with known estrogenic activity. LRE and LRP are both complex mixtures containing ISL as well as other components.

Working memory was assessed in the DSA study. Middle-aged female rats were ovariectomized (OVX) and exposed to ISL (0, 6, 12, or 24 mg) once per day through pellets.

Exposure to ISL did not affect DSA performance at any of the three doses tested. Estrogens have been shown to impair performance on the DSA task in previous studies. Spatial object location memory was assessed in three separate studies using the MCOL task. Object recognition memory was assessed using the DOR task. In the MCOL and DOR studies, young-adult female rats were

OVX and exposed to ISL, LRE or LRP continuously through the diet at concentrations of 0.075%,

0.5% and 5% respectively for three weeks. Estradiol groups were also included in each MCOL

iii and DOR study. The effects of these compounds in animals fed high fat (44.8% kcal from fat) or low fat (17.2% kcal from fat) diets were also assessed. The high fat diet is more similar to the

Western diet and was included to more closely model the human population consuming botanical estrogen supplements. In the MCOL studies, ISL and LRE were found to mimic the effects of estradiol and improve performance on the MCOL task in young-adult OVX rats. LRP did not affect performance on the MCOL task. There was no effect of dietary fat and no interaction between dietary fat and exposure to the botanicals or estradiol in the MCOL studies, thus diet was not included as a factor in the DOR study. In the DOR study, estradiol impaired performance on the task, replicating previous findings. Exposure to ISL, LRE or LRP did not affect performance on the DOR task.

Because of the significant cognitive effects of ISL and LRE seen on the hippocampus-sensitive

MCOL task, gene and protein expression of several estrogen-sensitive targets known to be involved in cognition in the hippocampus were also assessed. RT-qPCR was used to measure gene expression of Bdnf, Ntkr2, Dlg4, Gria1, Gria2, Grin1, Grin2B, Esr1 and Esr2. There were no significant changes in the expression of any of the chosen genes with exposure to the botanical compounds or estradiol. However, there was a trend for reduced expression of three genes.

Western blotting was used to measure protein expression of these genes: PSD-95, GluR1, and

GluR2. ISL, LRE, LRP and estradiol failed to modulate levels of those proteins. These findings suggest that ISL and LRE may, like other estrogens, enhance performance on some hippocampus- sensitive tasks in young-adult OVX rats, while potentially avoiding some of the deleterious cognitive effects of some other estrogens tested to date.

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my advisor Dr. Susan Schantz. Throughout my time in graduate school, she has struck a wonderful balance of providing guidance and allowing me to develop my skills as an independent scientist. Her mentorship has helped to make my time in graduate school the happiest and most fulfilling of my life so far. I would also like to thank my former lab members, Dr. Steven Neese, Dr. Suren Bandara and Dr. Supida Monaikul, along with our laboratory technician Mindy Howe. They were an enormous help to me, especially in my early graduate school years.

I greatly appreciate the additional feedback and guidance I have received from Dr. Donna

Korol. Much of the work detailed in this document would not have been possible without her previous work and continuous thoughtful input on my projects. I would also like to thank Dr.

Jodi Flaws for generously opening her lab to me. Not only did it give me the opportunity to learn new skills, but her mentorship will serve as a model for me for the rest of my scientific career.

The members of my thesis committee have also been instrumental in my progress through graduate school, and I would like to thank them for their feedback.

I feel very fortunate to be a part of the Neuroscience Program at the University of Illinois.

The flexibility and diversity of the program in addition to the collegial atmosphere created by the faculty and students make it an ideal graduate school environment.

In my personal life, I would like to acknowledge my partner Dr. Benjamin Zimmerman for his indispensable contributions to my life in science as well as outside of it. I would also like to thank the wonderful friends I have made here, as well as my unwaveringly supportive family.

Finally, I would like to thank my funding sources: support from the NIH (5P50AT006268-

05), a fellowship from the Neuroscience Program at the University of Illinois, as well as a

Toxicology Scholar award.

TABLE OF CONTENTS

CHAPTER 1: BACKGROUND AND SIGNIFICANCE...... 1

CHAPTER 2: SPECIFIC AIMS ...... 36

CHAPTER 3: THE EFFECTS OF THE BOTANICAL ESTROGEN ISOLIQUIRITIGENIN ON DELAYED SPATIAL ALTERNATION ...... 40

CHAPTER 4: LICORICE ROOT COMPONENTS MIMIC ESTROGENS IN AN OBJECT LOCATION TASK BUT NOT AN OBJECT RECOGNITION TASK...... 65

CHAPTER 5: POTENTIAL TARGETS OF LICORICE ROOT COMPONENTS IN THE HIPPOCAMPUS ...... 102

CHAPTER 6: CONCLUSIONS ...... 122

CHAPTER 1: BACKGROUND AND SIGNIFICANCE

Background on botanical estrogens

Botanical estrogens, also called phytoestrogens are non-steroidal plant compounds that can mimic estrogens in the body (Glazier and Bowman, 2001). These compounds are widely sold as dietary supplements, are available for consumers to buy over the counter and are not subjected to pre-market regulation. However, there is a dearth of research investigating the effects of many of these compounds.

The effects of soy, and its most estrogenic isoflavone, genistein, have been investigated in both human and animal studies (e.g Kritz-Silverstein et al., 2003; Kreijkamp-Kaspers et al., 2004;

Lephart et al., 2002; Neese et al., 2010b, Pisani et al., 2012, File et al., 2005). Some studies have found beneficial effects of soy isoflavone supplementation on cognition in humans (e.g Kritz-

Silverstein et al., 2003; File et al., 2005), whereas animal studies have reported either positive or negative effects depending on the type of task and the brain regions that are engaged. (e.g Pisani et al., 2012; Neese et al., 2010b). While soy and soy components have been investigated in several studies, many commercially available phytoestrogens, including licorice root components, have not been adequately investigated for their ability to impact cognition. The goal of this work was to investigate components of licorice root, a common ingredient in commercially available dietary supplements, for their ability to affect cognition in female rats in a low-estrogen state.

Components of licorice root

Many over the counter botanical estrogen supplements contain licorice root powder, which has been shown to exert estrogenic effects both in vitro and in vivo (Boonmuen et al., 2016; Maggiolini et al., 2002; Mersereau et al., 2008). Isoliquiritigenin (ISL) and liquiritigenin (LIQ) are two of the

2 primary bioactive compounds in licorice root (Boonmuen et al., 2016; Mersereau et al., 2008). ISL and LIQ readily convert back and forth and are found in equilibrium in the body after treatment with either compound (Simmler et al., 2013). Both compounds have a relative binding affinity for estrogen receptors (ERs) that is from 1,500 to 1,000 lower than that of estradiol (Mersereau et al.,

2008). In addition, both compounds are lipophilic and have relatively low molecular weight and thus likely cross into the brain through the blood brain barrier (Srihari et al., 2012). ISL binds directly to ERs in vitro in MCF7 and T-47D breast cancer cells and competes with estradiol for binding (Boonmuen et al., 2016; Miksicek et al, 1993; Tamir et al., 2001). A range of .1-1µM of

ISL is needed for a half-maximal response, compared to 0.2nM of estradiol (Miksicek et al., 1993).

Thus, at high concentrations, ISL is a potent binder of ERs. This effect would most likely be amplified in a condition where little endogenous estrogen is present, in which ISL would not be competing with endogenous ligands for the ERs.

ISL and LIQ have also been shown to have transcriptional activity through the ERs. ISL and

LIQ stimulate the activity of estrogen-responsive genes in MCF7 cells in vitro with a magnitude similar to that of estradiol, albeit at a much higher concentration (Boonmuen et al., 2016;

Maggiolini et al., 2002). This effect is blocked with the anti-estrogen ICI, suggesting that these compounds do indeed act through estrogen pathways (Maggiolini et al., 2002). In young-adult female rats, oral administration of licorice extract, which contains ISL and LIQ, causes increased creatine kinase activity, a marker for activation of estrogen responsive genes (Tamir et al., 2001).

These results suggest that ISL and LIQ not only bind to ERs, but also affect transcriptional activity through the ERs.

Estrogen receptors, their distribution and function in the brain

The two main estrogen receptors are estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). These two receptors are found in the nucleus, where they affect transcription, as well as in extra-nuclear sites, where they can initiate second messenger cascades (Segars and

Driggers, 2002). The distributions of ERα and ERβ show tissue and cell type specificity. ERα is more prevalent in the hypothalamus and the amygdala whereas ERβ is more prevalent in the hippocampus, prefrontal cortex and the thalamus (Gasbarri et al., 2009). Estradiol also binds with high affinity and exerts rapid actions through the membrane receptor GPER. GPER is most prevalent in the hippocampus, hypothalamus and midbrain (Prossnitz and Barton, 2011).

There are many mechanisms by which estrogens can affect brain function. Estrogens play a neuroprotective role in the brain, among many other functions. Estrogens are thought to offer neuroprotection through several mechanisms including antioxidant effects, interactions with growth factors in the brain, and increasing cerebral blood flow through vasodilation (Arevalo et al., 2014; Maki and Hogervorst, 2003). Estrogens also modulate acetylcholine synthesis, a neurotransmitter critical for learning and memory. Estrogens can increase levels of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis (Rocca et al., 2010; Sherwin,

2005).

There is some evidence to suggest that estrogens also cause an increase in spine density in the hippocampus via an NMDA dependent mechanism (Woolley and McEwen, 1994). Spines in the CA1 region of the hippocampus fluctuate naturally over the ovarian cycle in rats, with the highest density being during the point in the cycle when estrogen is highest (Milner et al., 2014;

Woolley and McEwen, 1994). Some studies have found that supplementation of estrogen to OVX rats also results in an increase in dendritic spines in the hippocampus compared to non-

4 supplemented control animals (for a review see Milner et al., 2014). However, some studies have failed to find estrogen-related changes in spine density (e.g. Markham et al., 2005), thus more research is needed. It should be noted that changes in NMDA receptor concentrations have been found in the hippocampus in the absence of changes in spine density. For example, Adams et al.

(2001) found that in aged OVX rats, while spine density in the hippocampus did not change in response to estrogen supplementation, the NR1 subunit of the NMDA receptor did increase compared to aged OVX control rats. These results indicate that changes in NMDA receptor concentrations and changes in spine density do not always coincide.

Estrogens and cognition

Estrogens and cognition, studies using young or young-adult rodents

Previous literature shows that estrogens can affect cognition in rodent models. However, the relationship is a complicated one. The effects of estrogens on cognition can vary widely depending on a variety of factors including the cognitive task and the brain areas engaged by the task, the dose of estrogen, the form of estrogen, the age of the animals, as well as the pattern and timing of administration.

Some studies have found that in young or young-adult ovariectomized (OVX) rats, supplementation with estradiol leads to improvement on hippocampus-sensitive tasks. For example, young adult OVX rats implanted with silastic capsules containing 25% estradiol and tested on an eight-arm radial maze with all arms baited were found to perform better than OVX vehicle control rats (Daniel et al., 1997). In another study, young adult OVX rats were implanted with capsules containing 5% estradiol and were tested on an eight-arm radial maze with all arms

5 baited beginning either 3 or 12 days after hormone supplementation began (Luine et al., 1998).

Blood levels of estradiol were between 17 and 32 pg/ml, approximately the levels found during proestrus (Brown et al., 1990). Interestingly, choice accuracy improved relative to vehicle controls in rats that had received estradiol for 12 days, but not in rats that received estradiol for only 3 days prior to testing, highlighting that the effects of estradiol can differ based on length of hormone supplementation. Similarly, OVX adult female rats injected with estradiol every 4 days for 10 weeks at a dose of 4 μg/kg or 40 μg/kg starting one week after OVX performed better on a Morris water maze than OVX control rats (El Bakri et al., 2004).

However, contrary to the above findings, it has been reported that the presence of ovarian hormones can instead impair acquisition of some hippocampal tasks. For example, in a study with male and female rats tested on a hippocampus-dependent spatial-contextual conditioning task, females tested during proestrus showed less spatial-contextual conditioning than either females tested in estrus or males (Markus and Zecevic, 1997). Similarly, on a Morris water maze, young

OVX females outperformed gonadally intact females, suggesting that the presence of ovarian hormones impaired acquisition of the Morris water maze (Daniel et al., 1999). However, as part of the same study, the rats were tested on a spatial radial arm maze task and the opposite effect was found, that gonadally intact females outperformed OVX females. This discrepancy could be due to differing conditions between the tasks such as the stress level and whether the task was food-reward motivated. Another study found that in normally cycling adult rats, females in estrus outperformed females in proestrus in the Morris water maze (Warren and Juraska, 1997). This is contrary to what the authors hypothesized would happen, given that estradiol levels are significantly higher during proestrus than during estrus. It is possible that this was due to the fact that in normally cycling rats, both estradiol and progesterone are elevated during proestrus, and

6 that the effects of progesterone or the interaction of progesterone with estradiol affect cognition differently than estradiol alone. To investigate this idea, Chesler and Juraska (2000) tested OVX adult rats on a Morris water maze following treatment with estrogen, progesterone, or both. They found that rats treated with estrogen plus progesterone were impaired on the task relative to control rats, whereas rats treated with either estradiol or progesterone alone were not. These findings support the idea that estradiol may not facilitate hippocampal learning if progesterone is also present. However, a later study found that in young normally cycling mice, mice in proestrus or metestrus performed better than rats in estrus on a single day Morris water maze task (Frick and

Berger Sweeney, 2001). It should be noted, however, that mice may rely less on spatial strategies to find the hidden platform in the Morris water maze than do rats, for whom the task was originally designed (Frick et al., 2000).

Some studies have also investigated the role of estrogens on non-hippocampal tasks in young-adult animals, although there are fewer of these than studies investigating the effects of estrogens on hippocampal function. Previous work has investigated the effects of estradiol supplementation on the delayed spatial alternation (DSA) operant task as well as a differential reinforcement of low rates of responding (DRL) operant task. DSA is a measure of working memory and is thought to primarily engage the prefrontal cortex rather than the hippocampus at delays of less than 15 seconds (Maruki et al., 2001). The DRL task is a measure of response inhibition and also engages the prefrontal cortex (Broerson and Uylings, 1999; Narayanan and

Laubach, 2006). Young-adult OVX rats implanted with a silastic capsule containing 10% estradiol

(representing a high physiological level) showed a deficit in DSA as well as DRL performance compared to OVX rats given cholesterol vehicle only (Wang et al., 2008). This study also included a sham OVX group. Contrary to the other findings of this study, sham operated rats, though they

7 had higher levels of ovarian hormones than OVX rats, did not differ significantly from OVX rats in either the DSA or the DRL tasks. Again, the presence of progesterone in cycling rats could have influenced the effects of estradiol on this task.

Other studies have found a different pattern of results on pre-frontal cortex sensitive tasks.

In a delayed alternation T-maze task, a test of working memory (Chisholm and Juraska, 2012;

Maruki et al., 2001), OVX adult rats receiving a low physiological dose of estradiol benzoate via subcutaneous injection 2 hours before the task showed reduced working memory errors at a short delay of 10 seconds. In contrast, high physiological and supraphysiological doses of estradiol benzoate did not affect performance. It should be noted however that this acute estradiol exposure paradigm differs significantly from the chronic exposure paradigm used in the DSA study discussed above (Wang et al., 2008). At a longer delay (40 seconds), the two higher doses of estradiol significantly increased working memory errors, but the low dose did not. While high doses of estradiol did impair performance on the task at the longer delay, the longer delay is likely also reliant on intact hippocampal function, not just prefrontal cortex function (Maruki et al., 2001;

Sloan et al., 2006). While the delayed alternation T-maze task, like the operant DSA task, is prefrontal cortex-sensitive, the two tasks likely differ in the degree to which the hippocampus is involved. Hippocampus lesioned rats are impaired on both spontaneous as well as forced alternation in a T-maze (Lalonde, 2002). However, hippocampal lesions do not seem to affect performance on operant working memory tasks at short delays of less than 20 seconds (Maruki et al., 2001; Sloan et al., 2006).

Some studies have investigated both a hippocampus-sensitive task and a striatum-sensitive task in similar paradigms. These studies are useful in comparing the effects of estrogens on different brain systems, given that several factors like the dose of estradiol used, the pattern of

8 administration as well as aspects of the task environment are held constant. Once such paired paradigm is the place or response learning version of the plus maze (e.g Korol and Kolo, 2002).

In the hippocampus-sensitive place version of the task, rats must use extra-maze cues to learn to reach the static goal arm. For the striatum-sensitive response version of the task, extra-maze visual cues are absent and the rat must learn to make an egocentric body turn to reach the food reward.

Young-adult OVX rats injected with 10μg (Korol and Kolo, 2002) or 4.5 μg/kg (Pisani et al., 2012) of estradiol 48 and 24 hours before testing performed better on the place version of the task than

OVX control rats. In contrast, OVX control rats performed better than estradiol-supplemented rats on the response version of the task. These results suggest that estrogens might differentially modulate different memory systems in the brain, and supports the finding that estrogens act upon learning in a task-dependent manner. Similarly, OVX young-adult female rats implanted with a

60-day release pellet (IRA) that released 45–80 pg/ml of estradiol performed better on a place- learning version of the eight-arm radial maze task than OVX control rats. Conversely, OVX control rats learned the response version of the maze faster than estradiol supplemented rats (Davis et al., 2005).

Thus, the literature on the effects of estradiol on cognition in young and young-adult rats is mixed. Results vary based on several factors including the cognitive task and the brain areas engaged by the task, the length and method of hormone supplementation as well as whether estrogen is present alone or with progesterone.

Estrogens and cognition, studies using middle-aged or aged rodents

The above studies investigated the effects of estradiol on cognition in young or young- adult rodents. Studies also have found beneficial effects of estrogen supplementation in middle-

9 aged or aged rodents on tasks that engage the hippocampus. Middle-aged (13 month old) OVX rats receiving estradiol or estradiol plus progesterone performed better on a spatial delayed match to position task (Gibbs et al., 2000). Estradiol treated rats were implanted subcutaneously with a silastic capsule containing 25% estradiol, which led to circulating levels of 15-25 pg/ml serum estradiol. Estradiol plus progesterone treated rats received weekly injections which produced mean circulating levels of 50 pg/ml estradiol and 10 ng/ml progesterone. Interestingly, hormone supplementation was only effective if it was started at the time of OVX, or three months later, but not if supplementation started ten months after OVX (Gibbs et al., 2000). In another study, middle- aged OVX rats were given estradiol through the drinking water in either a chronic or cyclic pattern of administration and tested on a Morris water maze. The exposure was approximately

47μg/kg/day, which led to blood levels within the physiological range. No differences were found between groups for the first three days of testing. However, on the fourth and final day, rats chronically supplemented with estradiol performed better than cyclically supplemented rats as well as OVX control rats (Lowry et al., 2010). Another study investigated the role of ovarian hormones on performance in a Morris water maze in aged (22-24 month old) rats (Warren and Juraska, 2000).

Females in persistent estrus outperformed pseudopregnant females. Persistent estrus is associated with moderate levels of estrogen and low levels of progesterone, while the pseudopregnant state is associated with moderate levels of estradiol as well as progesterone (Huang et al., 1978; Warren and Juraska, 2000). It is possible that elevated levels of progesterone in addition to estradiol may have negated some of estradiol’s enhancing effects in the pseudopregant rats. However, Markham et al. (2002) found that aged (16-month-old) OVX rats supplemented with estradiol and progesterone outperformed OVX control rats on a Morris water maze. Rats that received either acute estrogen replacement for two days (16.67 μg/kg 48 and 24 hours before testing), chronic

10 estrogen replacement for 28 days (silastic capsule containing 25% estradiol), or chronic estrogen plus progesterone replacement for 28 days (one silastic capsule containing 25% estradiol and 75% progesterone, another silastic capsule containing 100% progesterone) outperformed OVX control rats. Thus the literature on estrogen as well as estrogen plus progesterone supplementation to middle-aged or aged OVX rodents is mixed, and requires further research.

The effects of estradiol supplementation on a prefrontally-mediated operant DSA task have been assessed in young, middle-aged and old OVX rats (Wang et al., 2009). The middle-aged and old rats were retired breeders. Rats were implanted with a silastic capsule containing 5% estradiol,

10% estradiol or cholesterol. Chronic estradiol supplementation resulted in a deficit in DSA performance in all the tested age groups compared to age matched rats given cholesterol vehicle only. In a follow up experiment, middle-aged retired breeder rats were OVX and given either the

ERα agnonist propyl pyrazole triol (PPT), the ERβ agonist diarylpropionitrile (DPN) (0.02, 0.08, or 0.20 mg/kg/day), 10% estradiol in a silastic capsule or oil control and tested on the DSA task.

This resulted in serum estradiol levels of 20.82 ± 3.41 pg/ml. The estradiol group was impaired on the DSA task compared to oil control rats, replicating Wang et al., 2009 (Neese et al., 2010a). The study also found that a low dose of DPN impaired performance, but that PPT did not, suggesting that the estradiol induced deficit in DSA performance may be ERβ mediated (Neese et al., 2010a).

In contrast, Chisholm and Juraska (2012) found that in a delayed alternation T-maze task, chronic or cyclic administration of estradiol through drinking water to middle-aged OVX rats did not affect performance on the delay portion of the task (although animals receiving both estradiol and the synthetic progesterone medroxyprogesterone acetate acquired the task more quickly than control rats). Estradiol levels in Chisholm and Juraska (2012) were likely similar to those in Neese et al.

(2010a). However, as discussed previously, it is possible that performance on the delayed

11 alternation T-maze task relies more on the hippocampus than performance on the operant DSA task (Lalonde, 2002).

In summary, just as for young-adult rodents, the literature on the effects of estradiol on cognition in middle-aged and aged rodents is mixed and results can vary due to several factors.

These include the factors listed in the previous section, as well as the phase of reproductive senescence (i.e. persistent estrus vs. pseudopregnant).

Botanical estrogens, studies using young-adult rodents

While several studies have been conducted to investigate the effects of estrogens on cognition in rodent models, there are far fewer studies investigating the effects of botanical estrogens on cognition. A few studies have investigated the effects of genistein, a soy isoflavone, on young-adult rats. Young-adult OVX rats showed improved place-learning and impaired response-learning in a plus maze after oral supplementation with genistein. This pattern was similar to that with estradiol supplementation (Pisani et al., 2012). Genistein binds selectively to

ERβ over ERα (Jiang et al., 2013). Genistein was given orally at a dosage of 4.4 mg over 2 days prior to testing, to mimic blood levels seen in women taking supplements containing this soy isoflavone (Pisani et al., 2012).

The effects of soy compounds on cognition have also been tested in an ischemia model.

Soy extract at doses of 20 or 60 mg per day partially ameliorated an ischemia-induced impairment on the Morris water maze in adult OVX female rats (Vafaee, 2014). This mimicked the effect seen with estradiol (Vafaee, 2014). In a study investigating the effects of a lifelong diet high in soy phytoestrogens, male and female rats were given either a diet containing 600 μg of soy phytoestrogens/gram of diet or a phytoestrogen-free diet. Rats were tested in a radial arm maze,

12 first with eight-arms baited (working memory task), and then with four-arms baited (reference memory task). In both tasks the high phytoestrogen diet improved maze performance in females, while hampering it in males (Lund et al., 2001).

There are very few studies in the literature assessing the effects of licorice components on cognition. The effects of LIQ on amyloid- β-peptide neuropathy in rats, a model of Alzheimer’s disease, has been investigated in male and female rats. The rats were tested on the Morris water maze after injections of 0, 2.3, 7.0, or 21.0 mg/kg/day of LIQ. All three doses of LIQ partially rescued behavioral impairment on the Morris water maze task as well as attenuated neuronal loss in the hippocampus. LIQ administration also reduced notch-2 mRNA and protein levels compared to vehicle control rats, providing a possible mechanism for the partially rescued behavior in rats given LIQ (Liu et al., 2010).

Botanical estrogens, studies using middle-aged or aged rodents

Previous work has investigated the effects of genistein as well as S-equol, a metabolite of the soy isoflavone daidzein that is made in the mammalian gut, in middle-aged and old rats. The effects of oral genistein exposure (162 or 323 μg/kg/day) were tested in young, middle-aged and old OVX rats using operant DSA, DRL and reversal learning tasks (Neese et al., 2010b). On the

DSA task, the old rats receiving the higher dose of genistein performed worse than both the high dose young and middle-aged groups. On the DRL task, the high dose of genistein resulted in a marginally significant impairment in the ratio of reinforced to non-reinforced lever presses across all age groups. No effects of genistein were found in the reversal learning task (Neese et al.,

2010b). A later study found that unlike genistein, treatment with S-equol (0, 10,000 or 19,000

μg/kg/day orally) to middle-aged OVX rats had no effect on the DSA task or a hippocampus-

13 sensitive place-learning task. S-equol binds selectively to ERβ with an affinity similar to that of genistein but has lower transcriptional potency than genistein (Muthyala et al., 2004).

The effects of soy isoflavones on an eight-arms baited radial arm maze have also been investigated. OVX retired breeder female rats were given oral doses of 0, 72, or 144 mg/1,800 calories soy phytoestrogens or estradiol. These doses fall within the range of those taken by humans consuming soy supplements. Oral administration of estradiol or soy phytoestrogens resulted in a dose-dependent improvement in performance on the radial arm maze compared to

OVX control rats (Pan et al., 2000).

Estrogens and cognition, human studies

Several studies have also been conducted to investigate the effects of estrogens on cognition in humans. Estrogens may serve a protective role in the brain. Women with high serum concentrations of non-protein-bound and bioavailable estradiol, but not testosterone, were less likely to develop cognitive impairment than women with low concentrations (Yaffe et al., 2000).

This finding supports the hypothesis that higher concentrations of endogenous estrogens prevent cognitive decline (Yaffe et al., 2000). However, similar to studies in rodents, studies investigating the effects of estrogens on cognition in humans have yielded varying and inconsistent results.

When the WHI studies found an increased incidence of some cancers and stroke as well as some evidence of cognitive impairment in women receiving HRT, many women were driven away from it. In WHIMS, the memory component of the Women’s Health Initiative (WHI) study, a large randomized trial in post-menopausal women, women receiving estrogen or estrogen plus progesterone therapy were found to have an increased risk of dementia and cognitive decline

(Craig et al., 2005). The WHI studies have since been criticized for beginning hormone

14 replacement therapy well after the onset of menopause in most of their participants, a factor we now know to be important in determining the health effects of estradiol supplementation (for a review, see Rocca et al., 2010).

Many studies, including some meta-reviews have shown apparent cognitive benefits of

HRT. In a comparison of menopausal women receiving HRT to women with no previous HRT, there was a relative deficit on several measures of executive function including working memory and inhibition of inappropriate responses in the no HRT group (Keenan et al., 2001). A 2001 meta- analysis found that women who were symptomatic from menopause, typically women reporting somatic complaints, showed improvements in verbal memory, vigilance, reasoning, and motor speed with HRT (LeBlanc et al., 2001). That meta-analysis also found a decreased risk of dementia with HRT from observational studies. However, the authors point out that for several of the studies reviewed, there was not enough information to control for some important factors such as progestin use, type and dose of estrogen taken and the duration of HRT. A 2003 review of 27 studies found that most (21 of 27) found evidence of a cognitive benefit in at least one cognitive domain with

HRT. Only one study found evidence for a negative cognitive effect of HRT (Maki and

Hogervorst, 2003). The authors did state, however, that this could be influenced by positive publication bias.

There are some studies that have found no effect of HRT on cognition, or a negative effect

(e.g Barrett-Connor and Kritz-Silverstein, 1993; File et al., 2002; Matthews et al., 1999). For example, one study found that women receiving HRT for 10 years had significantly worse long term episodic memory as well as worse mental flexibility as measured by a rule reversal task compared to women not receiving HRT. Women were matched for age, IQ, socio-economic status, years of secondary education, and occupation (File et al., 2002). However, that study only followed

15 women who had undergone surgical menopause, and some literature suggests outcomes from surgical menopause are different from those of natural menopause (e.g Chisholm and Juraska,

2013). Additionally, the participants had undergone surgical menopause about ten years before the start of HRT. Many studies in animals and humans have indicated that the time between the loss of ovarian hormones and the beginning of hormone replacement therapy is an important factor in determining the outcome of HRT (e.g Chisholm and Juraska, 2013; Rocca et al., 2010).

Many factors could account for the differences in the outcomes across HRT studies. These include the form of HRT used (e.g estrogen vs. estrogen plus progesterone, synthetic vs. natural hormone), the types of statistical correction for “healthy user bias”, the length of hormone deprivation after menopause, and the types of tasks being administered to measure cognition.

Botanical estrogens, human studies

There are also a small number of studies investigating the effects of botanical estrogens on cognitive function in humans. In a 6-month, double-blind, randomized, placebo-controlled clinical trial, post-menopausal women who received soy isoflavones (110 mg/day) performed significantly better on a category fluency task than women receiving placebo. No effects were found on visuomotor ability or logical memory and recall tasks (Kritz-Silverstein et al., 2003). Women who were in good health, were postmenopausal at least 2 years, and were not using estrogen replacement therapy were included in that study (Kritz-Silverstein et al., 2003). In a double-blind, placebo-matched parallel groups study of 50 postmenopausal women, participants receiving the soy supplement Novasoy (60 mg total isoflavone equivalents per day) performed better than the placebo group on non-verbal short-term memory. Novasoy also improved performance on a mental

16 flexibility task and a planning ability task, two tests of frontal lobe function. No effects of Novasoy on long-term memory, category generation, or sustained attention were seen (File et al., 2005).

High fat and low fat diet

The typical Western diet is high in fat (Carrera-Bastos et al., 2011) and there is interest in whether high fat diets can negatively impact the same cognitive functions that are impacted by estrogens. Specifically, the typical Western diet is considerably higher in saturated fat than a typical laboratory rodent diet. The average Western diet includes about 15% of calories from saturated fat, approximately the level contained in the high fat diet used in the studies described in this thesis (German and Dillard, 2004; Dietary Guidelines for Americans, 8th Edition). The average rodent diet contains only around 3% of calories from saturated fat (Winocur and Greenwood,

2005). Given that the standard rodent diet is not a good representation of the average diet in the

Western world, the inclusion of high fat diet groups in my work allowed me to investigate whether

ISL, LRE and LRP have a different effect on cognition if combined with a diet more representative of the Western diet.

Several studies have shown that a high fat diet can impair cognition (for a review see

Freeman et al., 2014). Studies conducted by Winocur and Greenwood found that male adult rats fed a diet high in saturated fat were impaired on a hippocampus-dependent radial arm maze task as well as a prefrontal cortex-dependent operant delayed alternation task compared to rats fed a calorie matched diet containing unsaturated soybean oil (Winocur and Greenwood, 2005).

Cognitive impairments have even been reported following a short-term exposure to a high fat diet.

Murray et al. (2009) tested male adult rats on an eight-arm radial maze with all arms baited after nine days of exposure to a high fat diet. They found that even that short exposure was sufficient to

17 increase working memory errors in rats fed a high fat diet. However, most studies investigating the effects of a high fat diet in rodent models used only males. In the few studies investigating both males and females, some have found sex differences in response to a high fat diet in peripheral metabolism, performance on a contextual fear conditioning task, as well as the magnitude of long- term potentiation (Hwang et al., 2010; Underwood and Thompson, 2016), highlighting the need to investigate the effects of a high fat diet in females as well as males.

Considerations for the present study

As discussed above, phytoestrogens have the potential to impact cognition. Given that these compounds are available over the counter with no premarket regulation, and the fact that women are already consuming these compounds, it is important to investigate the effects these compounds can have on cognition. The goals for this research were to investigate licorice root components for their potential to affect cognition in an OVX rat model. Important considerations for designing the studies are discussed below.

Licorice root components

While soy isoflavones have been investigated in several studies, licorice root components have not been investigated nearly as thoroughly, despite the fact that they are in widespread use in dietary supplements (Geller and Studee, 2005). Licorice root powder has been shown to exert estrogenic effects both in vitro and in vivo, with ISL and LIQ being two of the most active estrogenic compounds found in licorice root (Boonmuen et al., 2016; Mersereau et al., 2008). ISL and its active metabolite LIQ readily convert back and forth in vivo and are found in equilibrium in the body after treatment with either compound (Simmler et al., 2013).

As discussed above, both estrogens and phytoestrogens have the potential to affect cognition in animal models and humans. For this reason, it is important to investigate the effects of licorice root and its components for their potential to affect cognition. I investigated isoliquiritigenin (ISL), a methanol extract of licorice root (LRE), and licorice root powder (LRP).

I chose to investigate LRE and LRP because those are the forms typically found in dietary supplements. I chose to investigate ISL because it is a pure compound that is one of the most active estrogenic compounds in licorice root (Boonmuen et al., 2016; Mersereau et al., 2008).

OVX rat model

My goals were to investigate the effects of the licorice root components on aspects of cognition in a low estrogen state. In the DSA study I investigated the effects of ISL on an operant working memory task. In the MCOL and DOR studies I investigated the effects of ISL, LRE and

LRP on a spatial object placement task and an object recognition task respectively. I used OVX rats to control endogenous estrogen exposure and to investigate the effects of licorice root components on cognition in the absence of ovarian hormones, similar to the hormonal environment found post-menopause in human females.

In the DSA study, I used middle-aged OVX rats. Our lab has extensive experience using middle-aged rats in this paradigm and has shown that estradiol, the ERβ agonist DPN and the soy phytoestrogen genistein all impair performance on the operant DSA task in OVX middle-aged rats

(Neese et al., 2010a; Neese et al., 2010b; Wang et al., 2009). In the MCOL and DOR studies, I used young-adult OVX rats. I used rats of this age as a starting point due to the fact that we had never used the MCOL or DOR tasks in our lab before and previous studies using these tasks to investigate the impact of estrogens were all done in young-adult rats (Korol and Pisani, 2015).

Behavioral testing

In the DSA study I used an operant DSA task to investigate the effects of ISL on working memory in a middle-aged OVX rat model. As discussed above, previous literature suggests that estrogen supplementation to OVX rats impairs performance on this operant DSA task (Neese et al., 2010a; Wang et al., 2008; Wang et al., 2009). Additionally, Neese et al. (2010a) found that in middle-aged OVX rats the ERβ agonist DPN impaired performance on the DSA task, but that the

ERα agonist PPT did not, suggesting that the estradiol induced deficit in DSA performance may be ERβ-mediated. Previous literature has also shown that ISL has estrogenic activity and is ERβ selective (Boonmuen et al., 2016; Mersereau et al., 2008; Maggiolini et al., 2002). Therefore, I wanted to investigate the effects of ISL on the DSA task.

Previous work suggests that operant based working memory tasks such as delayed matching to sample or delayed alternation do not require the hippocampus at delays less than around 20 seconds (Maruki et al., 2001; Sloan et al., 2006). Most work on the effects of estrogens on cognition have focused on hippocampus-sensitive tasks, while relatively little work exists on tasks that are largely independent of hippocampal function but rely on the prefrontal cortex. Thus, in Chapter 3, I utilized the DSA task to investigate the effects of ISL on a prefrontal cortex-mediated cognitive outcome.

In Chapter 4, I used the hippocampus-sensitive metric change in object location (MCOL) task, and the striatum-sensitive double object recognition (DOR) task to investigate the effects of

ISL, LRE and LRP on cognition in an OVX rat model. As discussed above, estrogens can have differential effects on tasks that rely on different brain areas. Therefore, I tested the effects of ISL,

LRE and LRP on two tasks that engage different memory systems and have been shown to be estrogen sensitive (Korol and Pisani, 2015).

Additionally, I chose these tasks because food restriction is not required for either task. In order to achieve continuous exposure as well as to introduce large enough quantities of the compounds through the diet, it was important to use tasks in which rats were fed ad libitum. The operant testing approach our group has used in much of our previous work requires food restriction, and thus, is problematic for studies in which exposure is through the diet. The MCOL and DOR tasks both utilize the rat’s natural inclination to explore novelty in the environment, in this case, in the form of novel locations of a pair of previously seen objects, or a novel pair of objects, differing from those explored initially.

The MCOL task was adapted from the lab of Dr. Raymond Kesner at the University of Utah.

The Kesner lab showed in 2008 that lesions to the dorsal hippocampus in rats significantly impair performance on this task (Goodrich-Hunsaker et al., 2008). They lesioned the dorsal dentate gyrus, dorsal CA3 or dorsal CA1 and found that lesions to all three subfields of the hippocampus led to significant deficits in performance on this task. The biggest deficit was found in rats with lesions to the dorsal dentate gyrus (Goodrich-Hunsaker et al., 2008).

Recent findings suggest that the MCOL task is hippocampus-sensitive and the DOR task is striatum-sensitive (Korol and Pisani, 2015). Additionally, hippocampal activation does not seem to be necessary for performing the DOR task and striatum activation does not seem to be necessary to perform the MCOL task (Korol and Pisani, 2015). A lidocaine infusion into the hippocampus of rats impaired performance on the MCOL task but not the DOR task. In contrast, a lidocaine infusion into the striatum of rats impaired performance on the DOR, but not the MCOL task (Korol and Pisani, 2015), suggesting a double dissociation between task and brain area.

Previous studies have also found that these tasks are sensitive to modulation by estrogens.

Young-adult female rats that were OVX showed significantly better performance on the MCOL

21 task when supplemented with 2 estradiol injections (45µg/kg each) at 48 and 24 hours prior to testing (Korol and Pisani, 2015). The same pattern of estradiol treatment impaired performance on the DOR task (Korol and Pisani, 2015).

The MCOL and DOR studies I conducted also included an estradiol group in each study that received the same pattern of estradiol exposure as rats in previous studies using these tasks

(e.g Korol and Pisani, 2015). I included estradiol groups to confirm previous findings of estrogen modulation on the MCOL and DOR tasks.

Potential molecular targets

As discussed above, ISL and LIQ have estrogenic activity (see “Components of licorice root”).

Given that the MCOL task is hippocampus-sensitive as well as estrogen-sensitive, I investigated targets that are present in the hippocampus, relevant to learning and memory, and are modulated by estrogens. While it has been shown that ISL can act through estrogen pathways, it has also been shown to have a variety of other functions. It has been shown to act as a vasorelaxant, an antioxidant, and anti-inflammatory agent and an anti-tumor agent (Zhan et al., 2006). It has also been shown to modulate GABA(B) receptors to inhibit the release of dopamine (Jang et al., 2008), and to be a sirtuin-activating compound (Alcaín and Villalba, 2009). However, since it has been shown to have activity at ERs and I found it to affect cognition on an estrogen sensitive hippocampally-mediated task (see Chapter 4), I chose targets in the hippocampus that are relevant to learning and memory and are modulated by estrogens as a starting point for mechanistic studies.

I used RT-qPCR to investigate changes in gene expression and Western blotting to investigate changes in protein levels. I chose to investigate gene expression of Bdnf, Ntkr2, Dlg4, Gria1,

Gria2, Grin1, Grin2B, Esr1 and Esr2 as my initial targets. I then investigated protein expression

22 of a subset of those targets (PSD-95, GluR1, and GluR2). These studies are discussed in detail in chapter 5.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors. It helps support the survival of existing neurons, and encourages the growth and differentiation of new neurons and synapses (Huang, 2001). BDNF is active in the hippocampus and vital for learning and memory (Huang, 2001). BDNF has also been shown to be modulated by estrogens. In adult, cycling rats, BDNF levels in the hippocampus are the highest on the day of proestrus and the morning of estrus compared to metestrus and OVX rats (Spencer et al., 2008). Levels of BDNF fluctuate in concert with fluctuations in pyramidal cell excitability in the hippocampus of rats over the course of the estrus cycle, and fluctuations are blocked by an inhibitor of tyrosine kinase receptors (Scharfman et al., 2003). OVX adult rats given estradiol have higher levels of BDNF mRNA and protein in the hippocampus compared to OVX control rats

(Spencer et al., 2008). Additionally, OVX retired breeder rats given either estradiol or oral soy isoflavone exposure had higher BDNF mRNA levels in the prefrontal cortex than OVX control rats (Pan et al., 1999). In that study, rats were given 150mg of total soy isoflavones per day, a dose chosen to mimic the exposure of humans consuming soy supplements.

TrkB, or tyrosine receptor kinase B is the primary receptor for BDNF, as well as other growth factors (Pan et al., 2010). Activation of TrkB is involved in spatial memory formation in rats (Mizuno et al., 2003). Several studies suggest that TrkB is also modulated by estrogens.

Immunoreactivity for phosphorylated TrkB (pTrkB) fluctuates across the estrus cycle in the hippocampus of adult female rats (Spencer et al., 2008). The authors found increased pTrkB in proestrus compared to diestrus. This fluctuation corresponds to fluctuations in BDNF protein expression, as might be expected (Spencer et al., 2008). In adult OVX mice given estradiol, levels

23 of pTrkB in the hippocampus increased 48 hours later but did not in OVX control rats (Spencer et al., 2008). Additionally, in both ERα and ERβ knockout mice, estrogen effects on pTrkB no longer exist, suggesting both receptors are involved in estrogen effects on TrkB.

PSD-95, or post-synaptic density protein 95, is a vital scaffolding protein. It is essential for the clustering of receptors, ion channels and associated signaling proteins in the synapse (Meyer et al., 2014; Sheng 2001). PSD-95 plays an important role in synaptic plasticity and the stabilization of synaptic changes during long term potentiation (Meyer et al., 2014; Sheng 2001).

Several studies have shown that PSD-95 is modulated by estrogens. In OVX rats, 48 hours of treatment with estradiol increases levels of PSD-95 in the CA1 region of the hippocampus

(Spencer et al., 2008). In cultured hippocampal neurons, supplementation with estradiol leads to increased levels of PSD-95 (Akama and McEwen, 2003). Levels of PSD-95 are highest in proestrus in naturally cycling rats (Spencer et al., 2008). In adult OVX female rats, estradiol, PPT and DPN all led to an increase in PSD-95 in the hippocampus (Waters et al., 2009).

GluR1 and GluR2 are both subunits of the ionotropic AMPA type glutamate receptor.

Levels of GluR1 and GluR2 change with the induction of long-term potentiation (LTP).

Immediately after LTP, GluR1 homomers are inserted into the synapse, which provides a new source of calcium and is a mechanism for the increase in synaptic strength post-LTP. Several minutes after the induction of LTP, GluR1 homomers are replaced with GluR1-GluR2 and GluR2-

GluR3 heteromers, which are involved in the later phase of LTP. An increase in GluR2 typically leads to reduced excitation (Kauer et al., 2006). There is evidence that hippocampal GluR1 and

GluR2 are modulated by estrogens. In adult OVX rats, estradiol, PPT or DPN led to an increase in

GluR1 in the hippocampus (Waters et al., 2009). Additionally, DPN increased expression of GluR2

24 in the hippocampus, but estradiol and PPT did not, suggesting that the estradiol-induced effect on

GluR2 might be ERβ mediated (Waters et al., 2009).

NR1 and NR2B are subunits of the ionotropic NMDA type glutamate receptor. The NMDA receptor plays a prominent role in hippocampal LTP as well as in spatial learning and memory

(Sun et al., 2010). Additionally, there is some evidence to suggest that estrogens cause an increase in spine density in the hippocampus via an NMDA receptor-dependent mechanism (e.g Woolley and McEwen, 1994). The NR1 subunit is obligatory in the NMDA receptor. There are four distinct isoforms of the NR2 receptor and the isoforms are expressed differentially in different tissues

(Ryan and Grant, 2009). Knocking out NR1 in the CA1 of mice results in impairment in contextual fear memory as well as taste memory (Li et al., 2007). A conditional knock out of NR2B in CA3 results in severely impaired LTP in that region (Akashi et al., 2009). There is evidence that these subunits are modulated by estrogens in the hippocampus. In adult OVX rats, ovariectomy reduced mRNA levels NR1 and NR2B, and estradiol supplementation prevented this decrease (Cyr et al.,

2001).

I chose to investigate Esr1 and Esr2 because ISL, LRE and LRP have activity at the estrogen receptors (Boonmuen et al., 2016; Maggiolini et al 2002; Mersereau et al, 2008).

Additionally, ISL, LRE or LRP could be modulating the other targets we selected through the ERα and ERβ receptors.

In summary, I chose the above targets because they are important for cognition in the hippocampus and are modulated by estrogens. ISL, LRE and LRP could be acting on these targets.

However, it is also possible that they are acting through other mechanisms and future studies may be needed to address these.

Conclusions

As reviewed above, estrogens can modulate cognition. Botanical estrogens can mimic estrogens in the body and thus also have the potential to affect cognition through estrogen pathways. In the DSA study described in Chapter 3, I investigated the effects of ISL on the prefrontal cortex-sensitive DSA task. In the MCOL and DOR studies described in Chapter 4, I investigated the effects of ISL, LRE and LRP on the hippocampus-sensitive MCOL task and the striatum-sensitive DOR task. Given that botanical estrogens, including the compounds I investigated in these studies (ISL, LRE and LRP), are available over the counter and are not subject to premarket regulation, it is important to investigate the effects these compounds may have on cognition. I used an OVX female rat model in order to investigate the effects of these compounds in a low estrogen state. The MCOL and DOR studies were done using OVX young-adult animals rather than middle-aged or aged animals, and are a first step in determining the effects of these compounds on cognition in the absence of endogenous estrogens. Given that botanical estrogens are often marketed to peri- and post-menopausal women, future studies using middle-aged animals could determine the effects of these compounds on cognition in that age group. A high fat diet group was also included for each exposure in the MCOL study in order to more accurately model the human population consuming these compounds.

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CHAPTER 2: SPECIFIC AIMS

It is known that estrogens can have effects on cognition and that these effects can vary based on several factors. As discussed in chapter 1, estrogens often improve performance on hippocampal tasks and impair performance on striatal and prefrontal tasks in young adult ovariectomized (OVX) rats, although there are several exceptions (see Chapter 1, Estrogens and cognition). The tasks I chose to evaluate the cognitive effects of licorice root components differ in terms of the brain regions that are critical for accurate performance. The delayed spatial alternation (DSA) task is prefrontal cortex-sensitive (Maruki et al., 2001). The metric change in object location (MCOL) task requires an intact hippocampus, whereas the double object recognition (DOR) task requires an intact striatum (Korol and Pisani, 2015; Tunur et al., 2012).

The DSA task is done in an automated operant chamber. The MCOL and DOR tasks are both object exploration tasks and are identical for the first three sessions, but importantly, vary in the final session such that the brain areas critical for accurate performance in the two tasks differs.

Licorice root was selected because it contains compounds including isoliquiritigenin (ISL) and liquiritigenin (LIQ) that have been shown to have estrogenic activity (Boonmuen et al., 2016;

Maggiolini et al 2002; Mersereau et al, 2008) and because very little research exists investigating its effects on cognition despite its widespread use in dietary supplements. In the DSA study, I investigated the effects of ISL on working memory. In the MCOL and DOR studies, I investigated the effects of ISL, a licorice root extract (LRE) and the whole licorice root powder (LRP) on object location and object recognition memory respectively. Since the MCOL and DOR tasks are identical until the final test trial, the effects of these botanicals in two distinct memory systems

(hippocampal and striatal) could be investigated and easily compared. Additionally, I was interested in whether a high fat diet would affect performance on these tasks and whether ISL,

LRE or LRP would interact with a high fat diet. The inclusion of the high fat diet groups also served to more accurately model the human population consuming licorice root compounds. Thus, in the MCOL study, I included both a high fat and a low fat group for each of the exposures. If dietary fat was shown to be an important factor, I planned to also include both low and high fat diet groups in the DOR study. However, no effects of dietary fat or interactions between dietary fat and the test compounds were found in the MCOL study, thus high fat diet groups were not included in the DOR study. An estradiol group was included in each of the experiments within the

MCOL and DOR studies to provide a replication of previous findings (Korol and Pisani, 2015;

Tunur et al., 2012) that estradiol improves performance on the hippocampus-sensitive MCOL task and impairs performance on the striatum-sensitive DOR task.

Aim 1: To determine whether ISL affects performance on the DSA task.

Middle-aged female Long-Evans rats were OVX and given one of three oral doses of ISL in sucrose pellets, or sucrose pellets alone. The rats were then tested on the operant DSA task.

Based on previous findings for estradiol and for other estrogenic compounds, it was hypothesized that ISL would lead to impaired performance on the DSA task in OVX middle- aged rats.

Aim 2: To determine whether components of licorice root (ISL, LRE, LRP) affect performance on the MCOL task and/or the DOR task.

Young-adult female Long-Evans rats were OVX and fed diets containing ISL, LRE or LRP. An estradiol group was also included in each experiment, as well as an OVX control. The rats were then tested on the MCOL task or the DOR task.

It was hypothesized that ISL, LRE, LRP and estradiol would lead to improved performance on the MCOL task and impaired performance on the DOR task in OVX young-adult rats.

Aim 3: To determine whether a high fat diet affects performance on the MCOL task and whether there is any interaction between exposure to ISL, LRE or LRP and a high fat diet.

Young-adult Long-Evans rats were fed either a high fat diet (44.8% kcal from fat) or a low fat diet (17.2% kcal from fat). Thus each exposure group (ISL, LRE, LRP, Estradiol, Control) was further divided into a high fat and a low fat group.

It was hypothesized that a high fat diet would impair performance on the MCOL task, and that this effect would be ameliorated or partially ameliorated by exposure to ISL, LRE and LRP.

Aim 4: To investigate the mechanism(s) of action for the observed behavioral effects in the

MCOL study.

The expression of the following genes was measured in the hippocampi of animals from the

MCOL study using RT-qPCR: Bdnf, Ntkr2, Dlg4, Gria1, Gria2, Grin1, Grin2B, Esr1 and Esr2.

These targets were chosen because they are involved in cognition in the hippocampus and are modulated by estrogen exposure. ISL, LRE and estradiol all improved task performance in the

MCOL study. Based on the results of the RT-qPCR, protein expression of a subset of the above targets (PSD-95, GluR1, GluR2) was investigated using Western blotting.

Based on the behavioral findings, it was hypothesized that mRNA and protein levels of these targets would be increased with exposure to ISL, LRE and estradiol compared to OVX control animals not given estradiol or any botanicals.

References

Korol, D. L., & Pisani, S. L. (2015). Estrogens and cognition: Friends or foes?: An evaluation of the opposing effects of estrogens on learning and memory. Hormones and behavior, 74, 105- 115.

Maruki, K., Izaki, Y., Hori, K., Nomura, M., & Yamauchi, T. (2001). Effects of rat ventral and dorsal hippocampus temporal inactivation on delayed alternation task. Brain research, 895(1-2), 273-276.

Tunur, T., Zendeli, L., & Korol, D. L. (2012). Nuances of pattern separation determine modulation by estradiol. In 42nd Annual meeting for the Society for Neuroscience, Society for Neuroscience.

Winocur, G., & Greenwood, C. E. (2005). Studies of the effects of high fat diets on cognitive function in a rat model. Neurobiology of aging, 26(1), 46-49.

CHAPTER 3: THE EFFECTS OF THE BOTANICAL ESTROGEN, ISOLIQUIRITIGENIN ON DELAYED SPATIAL ALTERNATION

Abstract Age-related declines in cognitive function can impair working memory, reduce speed of processing, and alter attentional resources. In particular, menopausal women may show an acceleration in the rate of cognitive decline as well as an increased vulnerability to brain diseases as estrogens may play a neuroprotective and neurotrophic role in the brain. To treat menopausal symptoms, many women turn to botanical estrogens that are promoted as a safe and natural alternative to traditional hormone replacement therapy. However, the majority of these compounds have not been systematically evaluated for efficacy and safety. The current study investigated the efficacy of the commercially available botanical estrogenic compound isoliquiritigenin (ISL) to alter performance on an operant working memory task, delayed spatial alternation (DSA). ISL is a compound found in licorice root that has been shown to have a wide range of effects on different biological systems, including estrogenic properties. This botanical is currently being used in over the counter dietary supplements. Middle-aged (12-month old) Long-

Evans female rats were ovariectomized and orally dosed with either 0 mg, 6 mg, 12 mg or 24 mg of ISL 60 minutes before testing on the DSA task. The DSA task required the rat to alternate its responses between two retractable levers in order to earn food rewards. Random delays of 0, 3, 6,

9 or 18 seconds were imposed between opportunities to press. ISL treatment failed to alter DSA performance. Previous work from our research group has found that estrogenic compounds, including 17β-estradiol and the botanical estrogen genistein impair performance on the DSA task. The goal of our botanical estrogens research is to find compounds that offer some of the

1 This chapter appeared in its entirety in Neurotoxicology and Teratology and is cited in this dissertation as “Kundu, Payel, et al. "The effects of the botanical estrogen, isoliquiritigenin on delayed spatial alternation." Neurotoxicology and teratology 66 (2018): 55-62. DOI: 10.1016/j.ntt.2018.02.001. This article is reprinted with the permission of the publisher.

beneficial effects of estrogen supplementation, without the harmful effects. This work suggests that ISL may not carry the cognitive risks associated with most other estrogenic compounds tested to date.

Introduction

Botanical estrogens are non-steroidal plant compounds that can mimic estrogen in the body

(Glazier and Bowman, 2001). These compounds are widely sold as dietary supplements despite a dearth of research on their effects (Mahady et al., 2003). Many over the counter botanical supplements contain licorice root powder (Geller and Studee, 2005). Isoliquiritigenin (ISL), and its active metabolite liquiritigenin (LIQ) are two of the primary bioactive compounds in licorice root and both compounds have been demonstrated to have estrogenic activity both in vivo and in vitro (Maggiolini et al 2002; Mersereau et al, 2008; Miksicek et al 1993).

There is a large body of research indicating that estrogens can affect cognition (Engler-

Chiurazzi et al., 2016; McEwen, 2001). However, the effects of estrogens on cognition can vary widely depending on a variety of factors including the cognitive task and the brain areas engaged during completion of the task. Specifically, the prefrontal cortex and the hippocampus are known to play important roles in working memory tasks (D'Esposito, 2007; Floresco et al, 1997). Most rodent tests of working memory have utilized maze paradigms, and these tests often involve the use of cue configurations together with relatively long delays between testing trials. Both of these factors likely engage the hippocampus during performance of the task (Maruki et al, 2001; Sloan et al, 2006; Wang and Cai, 2006). In contrast, operant based working memory tasks such as delayed matching to sample or delayed alternation that use short inter-trial delays of less than 20 seconds do not appear to require the hippocampus (Maruki et al, 2001; Sloan et al, 2006). Most work on

42 the effects of estrogens on learning and memory has focused on hippocampus-sensitive tasks, while relatively little work has focused on tasks that rely on the prefrontal cortex. Thus, we have utilized a delayed spatial alternation (DSA) operant task to investigate the effects of estrogens on a prefrontal cortex mediated aspect of learning and memory.

We have previously reported that ovariectomized (OVX) young (3 month old), middle- aged (12 month old) or old (18 month old) rats given physiological levels of 17β-estradiol were impaired on an operant DSA task relative to OVX vehicle control treated rats (Wang et al., 2008;

Wang et al, 2009). Wang et al. (2008) tested young (3 month old) OVX rats on the operant DSA task. Rats were implanted with a silastic capsule containing 17β-estradiol (serum level of

20.82 ± 3.41 pg/ml: Wang et al., 2009) or one containing cholesterol. This 17β-estradiol dose was chosen to mimic a high physiological level. We found that chronic 17β-estradiol supplementation resulted in a deficit in DSA performance compared to rats given cholesterol vehicle only. In a follow up study, Wang et al. (2009) tested young (3 month old), middle-aged (12 month old) and old (18 month old) OVX rats on the DSA task. Rats were implanted with a silastic capsule containing 10% 17β-estradiol or cholesterol. We found that chronic 17β-estradiol supplementation resulted in a deficit in DSA performance in all the tested age groups compared to age matched rats given cholesterol vehicle only. In order to further investigate the role of estrogens in modulating performance on the DSA task, middle aged (12 month old) rats were OVX and given either the

ERα agonist propyl pyrazole triol (PPT), the ERβ agonist diarylpropionitrile (DPN) (0.02, 0.08, or

0.20 mg/kg/day for both agonists) or oil control (Neese et al., 2010a). Another group received 10%

17β-estradiol in a silastic capsule to serve as a positive control. This study replicated the estradiol results from Wang et al. (2009), finding that the 17β-estradiol group was impaired on the DSA task compared to age-matched oil control groups. This study also revealed that a low dose of the

ERβ agonist DPN impaired performance, but that the ERα agonist PPT did not, suggesting that the

17β-estradiol induced deficit in DSA performance may be ERβ mediated (Neese et al., 2010a).

We have also investigated the effects of the botanical estrogens genistein and S-equol on

DSA performance. Genistein is an ERβ selective isoflavone found in soy. S-equol is a metabolite of the soy isoflavone daidzein and is also ERβ selective (Muthyala et al., 2004). Neese et al.

(2010b) tested the effects of oral genistein exposure in young (7 month old), middle-aged (16 month old) and old (22 month old) Long-Evans OVX rats on the DSA task. Rats were given genistein orally once daily at either a low dose (162 μg/kg/day) or a higher dose (323 μg/kg/day).

We found that the old rats receiving the higher dose of genistein performed worse than both the young and middle-aged groups given that dose. Neese et al. (2012) investigated the effects of three daily exposures to genistein (3.4 mg/kg each time) on DSA and DRL performance in middle-aged

(14 month old) OVX rats. This exposure paradigm was designed to keep serum genistein levels in the range of those found in humans consuming commercially available soy isoflavone supplements. We found that there was a trend for genistein treated rats to perform worse than sucrose control rats overall in DSA performance. Genistein treatment did not affect performance on the DRL task. Neese et al. (2014) also tested middle-aged (12-13 month old) OVX rats on the

DSA task after treatment with S-equol. S-equol binds selectively to ERβ with an affinity similar to that of genistein but has lower transcriptional potency than genistein (Muthyala et al., 2004). S- equol was given at 10,000 or 19,000 μg/kg/day orally. We found that S-equol did not affect performance on the DSA task.

In the present study, we investigated the effects of ISL on the same operant DSA task used to assess other estrogens in these previous studies. Both ISL and LIQ can bind to estrogen receptors and LIQ has a twenty-fold higher affinity for ERβ over ERα (Mersereau et al, 2008). However,

ISL and LIQ readily convert back and forth, and serum levels of the two compounds rapidly come to equilibrium after treatment with either compound (Simmler et al., 2013). Both compounds are lipophilic and have relatively low molecular weight and likely cross into the brain through the blood brain barrier (Srihari et al, 2012).

We chose to treat the rats with ISL, which is more readily available commercially. Similar to other estrogens, we hypothesized that treating OVX middle-aged (12 month old) rats with ISL would result in impairment on the operant DSA task. Dietary supplements containing ISL are already available for consumers to buy with no pre-market regulation. These supplements are marketed primarily to peri- and post-menopausal women, thus we used middle-aged OVX rats in order to model this population of consumers

Materials and methods

Animals and botanical exposure

A total of 64 middle-aged female Long-Evans rats were obtained from Harlan

(Indianapolis, IN). The rats were received in one cohort. All animals were maintained in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal

Care (AAALAC). Rats were housed in a temperature and humidity controlled room (22 °C, 40–

55% humidity) on a 12-h reverse light–dark cycle (lights off at 7:00 am). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of

Illinois at Urbana-Champaign and were in accordance with the guidelines of the Public Health

Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Care

45 and Use of Mammals in Neuroscience and Behavioral Research (NIH, 1986; Van Sluyters and

Obernier, 2004).

Rats were 12 month old retired breeders, and were assigned to one of four exposure groups (0,6,12 or 24 mg of ISL). The body weights of the rats ranged from 272 to 373 grams

(M=316, SD=22). Thus, the doses of ISL correspond to approximately 0, 19, 38, or 76 mg/kg body weight. There were 16 rats per group. Rats were pair-housed in standard plastic cages

(45 × 24 × 20 cm) with Beta Chip® bedding, and were allowed to acclimate to the vivarium for two weeks before ovariectomy. For the ovariectomy surgery, rats were anesthetized with isoflurane gas. Following removal of the ovaries, muscle and fat layers were individually sutured with silk suture thread (Fisher Scientific). The incision in the skin was then closed with stainless steel wound clips. A postsurgical injection of carprofen (5 mg/kg, s.c.) was administered for pain management. All animals were maintained on an AIN-93G soy-free diet (Harlan-Teklad,

Madison, WI) after surgery to avoid exposure to dietary estrogens. Water was available ad libitum. Beginning nine days after the OVX surgery, rats were food restricted to reduce them to

85% of their free-fed body weights. Each animal was weighed daily and given a ration of food

30 minutes after testing. The amount of food given was adjusted daily based on current weight in comparison to free feeding weight. Twelve days after food restriction began, ISL treatment and operant testing began. Rats were tested once per day, 6 days per week during the dark phase of the light cycle in a darkened testing room. One hour prior to testing, rats were given fruit punch flavored pellets (Test Diet, Richmond, IN) containing doses of roughly 0mg/kg/day,

19mg/kg/day, 38mg/kg/day, or 76mg/kg/day of ISL. Rats were dosed 7 days per week, including on days they were not tested.

Serum ISL and LIQ concentration analysis

Serum ISL and LIQ levels were measured in a pilot study prior to the current study (Figure

3.1). Six Long-Evans rats aged 9-12 months were OVX and fed an AIN soy-free diet for 4-5 weeks.

Rats were fed a dosing pellet containing 10mg of ISL and blood was drawn from a tail vein prior to dosing and again at 15 min, 30 min, 45 min, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after dosing. The mean dose of ISL used in the pilot study was between the lowest (6mg) and middle dose (12mg) used in the current study. This resulted in peak blood levels of total ISL and LIQ

(conjugated + unconjugated forms) of approximately 5μM and 1μM respectively (Figure 3.1A).

The peak aglycone levels of ISL and LIQ were approximately 0.5 μM and 0.01 μM respectively

(Figure 3.1B). Dosing in the current study was timed so that rats had peak blood levels of ISL at the time of testing. Thus, at the time of DSA testing, total blood levels of ISL were likely between

3μM and 10μM, depending on the dose group. Levels of total ILQ and LIQ were determined using LC/MS/MS following quantitative hydrolysis of conjugates by β- glucuronidese/arylsulfatase (H. pomatia; Sigma H-1, 2 units/assay) sing procedures previously described (Madak-Erdogan et al., 2016). Levels of aglycone ILQ and LIQ were determined without the enzymatic hydrolysis step.

Organ collection

At the time of sacrifice, liver and uterine horn weights were taken. The liver weight was divided by the body weight of the animal to derive a liver/body weight ratio. A higher liver/body weight ratio implies increased induction of liver enzymes, which is suggestive of hepatotoxicity

(Maronpot et al, 2010). It is important to look at measures of toxicity when testing new compounds. The uterine horn was also weighed. This was done because uterine proliferation is

47 suggestive of estrogenic activity, especially activity at estrogen receptor alpha (ERα)

(Matthiessen, 2013). The horn was removed and proximal fat and vasculature were trimmed off to yield accurate weight of the horn. The uterine horn was also measured in length using a ruler.

The weight of the uterine horn was divided by the length to yield a weight/length ratio (as described in Pisani et al, 2015).

Operant testing

Apparatus

Rats were behaviorally tested in standard automated operant chambers housed in sound- attenuated wooden boxes (Med Associates Inc., St. Albans, VT). The test chambers were

21.6 cm tall with a 29.2 cm × 24.8 cm stainless-steel grid floor that rested just above a tray filled with corn cob bedding. A pellet dispenser centered 2.5 cm above the floor on the operant panel dispensed 45 mg soy-free purified food pellets (Test Diet, Richmond, IN). Retractable response levers were located symmetrically on both sides of the pellet dispenser with a stimulus cue light above each lever. The levers were 5.7 cm from midline and 7.0 cm above the floor and the cue lights were located 5.7 cm above the levers. Each chamber also contained a Sonalert tone generator, a white noise generator, and a house light located on the back wall that remained off during testing.

Rats were fed a measured amount of food, sufficient to maintain 85% free-fed weight,

30 min after behavioral testing was completed each day. This feeding schedule was necessary to ensure that the animals were motivated to work for the food rewards used in the DSA test.

Response shaping

An autoshaping program was used to train rats to press the response levers (Widholm et al., 2004). At the beginning of the session, both response levers were extended into the operant chamber with white noise played continuously to mask extraneous noise. The cue light above the right response lever was illuminated for 15 seconds every 3 minutes according to a fixed-time 3- minute schedule (FT-3). After the cue light was turned off, a tone was played for 40ms and a single food pellet was provided at the same time. Presses to either lever at any time during the autoshaping session resulted in immediate reinforcement. The FT-3 schedule terminated when the animal had pressed a total of 10 times on either response lever during a session, at which point reinforcer delivery became contingent only on lever presses. Autoshaping test sessions terminated after 60 minutes elapsed or 100 reinforcers were delivered, whichever occurred first.

Criterion for this condition was set at 100 lever presses within a single session.

Lever press training

After autoshaping, all animals went through a continuous reinforcement schedule in which the reinforced lever alternated after the delivery of every fifth reinforcer. This was done to strengthen the lever-press response the animal learned during autoshaping and to prevent the animals from developing a preference for a particular side or lever. At the start of each session, either the right or left lever was extended into the chamber while the cue light above that lever lit up. Pressing the extended lever resulted in reinforcement in the form of a food pellet. After the animal had pressed the extended lever five times, the cue light was extinguished and the lever was retracted. This repeated with the opposite lever. This cycle of alternating levers terminated

49 after 100 reinforcers were received or 60 minutes had elapsed. Criterion for this condition was

100 reinforced lever presses for two or more consecutive sessions.

Cued alternation (CA)

Following lever press training, the rats were trained on a delayed spatial alternation

(DSA) task with a variable delay, as detailed in Widholm et al (2004). DSA was preceded by two training tasks. In the first, cued alternation (CA), the cue light indicated the correct lever on each trial, and the cue light alternated between the right and left levers. Rats were reinforced for pressing the lever opposite the one pressed on the previous trial, regardless of whether that trial was correct or incorrect. The rat’s response on the first trial of a session was always correct.

Each correct lever press resulted in reinforcement in the form of a food pellet, which was paired with a 40ms tone. Pressing the incorrect lever resulted in the retraction of the levers and the extinguishing of the cue-light. In between each trial, both levers were retracted and then immediately re-extended at the same time. There was no inter trial delay and no time limit to press a lever. Criterion for this condition was at least 60% correct cued lever presses within a session.

Non-cued alternation (NCA)

After CA, a non-cued spatial alternation task followed. In this condition, the cue light no longer indicated the correct lever, but correct responses still consisted of alternating between the left and right levers. Rats were reinforced for pressing the lever opposite the one pressed on the previous trial, regardless of whether that trial was correct or incorrect. The first trial of a session was always correct. Reinforcement was delivered in the form of a food pellet, which was paired with a 40ms tone. Pressing the incorrect lever resulted in the retraction of the levers and

50 immediate simultaneous re-extension of the levers to begin a new trial. There was no time limit to press a lever. Each animal was tested for 10 sessions in this condition.

Delayed spatial alternation (DSA)

The DSA task was the final phase of testing. In this condition, delays of 0, 3, 6, 9 or 18 seconds were imposed semi-randomly between alternation trials, with the stipulation that no particular delay was presented on more than three consecutive trials. There were 40 trials at each delay and a total of 200 trials per session. Rats were reinforced for pressing the lever opposite the one pressed on the previous trial, regardless of whether that trial was correct or incorrect.

Reinforcement was delivered in the form of a food pellet, which was paired with a 40ms tone.

Pressing the incorrect lever resulted in the retraction of the levers and the immediate beginning of the delay period. In between each trial, both levers were retracted and then either immediately

(0 second delay) or after 3, 6, 9 or 18 seconds they were re-extended. Both levers were retracted and then extended at the same time after the delay. Levers were not available during the delay.

There was no time limit to press a lever. Each animal was tested for 25 sessions.

Statistical analysis

The data were analyzed via ANOVA using Systat for Windows Version 13.1

(systatsoftware.com/products/systat/). Exposure was included as a between-subjects factor and significance was set at p<0.05. For autoshaping, the days to reach criterion served as the measure for learning. The data for autoshaping were analyzed using a one-way ANOVA with exposure as a between-subjects factor. For CA, the number of errors that occurred across all sessions and days to reach criterion both served as measures of learning. The data were analyzed

51 using a one-way ANOVA with exposure as a between-subjects factor for both measures. For

NCA, the proportion correct across the ten testing sessions was used as the measure of learning.

The data were analyzed using a 4 (exposure) × 10 (day) mixed ANOVA with day as a repeated measures factor. For DSA, the proportion correct was first combined into blocks of five sessions by averaging the proportion correct across sessions within each block. The proportion correct was then analyzed using a mixed 4 (exposure) × 5 (block) × 5 (delay) repeated measures

ANOVA with block (1–5) and delay (0, 3, 6, 9, and 18 s) serving as repeated measures factors. The latency to respond before a correct response, an incorrect response, and latency to respond overall was also analyzed. This was done using a repeated measures ANOVA with block as a repeated measures factor and exposure as a between-subjects factor. The organ weights were analyzed via one-way ANOVA with exposure as the between subjects factor.

Results

Autoshaping

The days to reach criterion was used as the measure of learning during this phase. There were no main effects of exposure on the days to reach criterion (F[3,60]=0.532, p=0.662, Figure

3.2).

Cued alternation (CA)

Both the number of total errors as well as the days to reach criterion were used as measures of learning during this phase. There were no main effects of exposure on either the total errors

(F[3,60]=0.115, p=0.951, Figure 3.3A) or the number of days to reach criterion (F[3,60]=0.182,

52 p=0.908, Figure 3.3B). There was no main effect of exposure on the latency to respond during the

CA phase (F[3,59]=1.917, p=0.137). There was also no effect of exposure on the latency to respond before a correct response (F[3,59]=1.662, p=0.185) or an incorrect response

(F[3,59]=1.794, p=0.158).

Non-cued alternation (NCA)

The proportion correct was used as a measure of learning in the NCA phase. There was no main effect of exposure on proportion correct during NCA (F[3,60]=1.284, p=0.288, Figure 3.4), and no interaction of exposure and day (F[19.262, 385.243]= 1.303, p=0.176). The analysis did reveal a significant effect of day as expected (F[6.421, 385.243]=103.423, p<0.001), showing that all exposure groups increased their proportion correct across days, an indication that learning occurred. There was no main effect of exposure on the latency to respond overall during the NCA phase (F[3,60]=2.045, p=0.117). There was also no effect of exposure on the latency to respond before a correct response (F[3,60]=2.154, p=0.103) or an incorrect response (F[3,60]=1.033, p=0.384).

Delayed spatial alternation (DSA)

The proportion correct was used as a measure of learning in the DSA task. There was no main effect of exposure on the proportion correct (F[3,60]=1.149, p=0.337, Figure 3.3A). There were trends for interactions between exposure and testing block (F[72.000, 117.000]=1.341, p=0.079) and exposure and delay (F[12, 177]=1.720, p=0.066), (Figures 3.5A and 3.5B). The lowest dose group (6mg ISL) had the lowest proportion correct across blocks of testing, although this was not a statistically significant difference. Across delays, all exposure groups were below

53 controls with this difference being clearest in the lowest dose group. However, simple main effects at each block as well as at each delay did not approach significance. There was no significant interaction between exposure, block and delay (F[54.673, 1093.458]=1.181, p=0.177). The analysis did reveal a significant effect of block (F[25.482, 37.00]=25.482, p<0.001) indicating that all exposure groups increased their proportion correct across block, an indication that learning occurred. There was also a significant effect of delay (F[4, 57]=908.8, p<0.001) indicating that all groups had lower proportions correct as the length of the delay increased (Figure 3.5B).

Latency to respond during DSA

There was no main effect of exposure on the latency to respond overall during the DSA task (F[3,60]=1.916, p=0.137). There was a significant effect of block on the latency to respond overall showing that the latency to respond tended to decrease across blocks of testing

(F[4,240]=19.359, p<0.001). In addition, there was also no effect of exposure on the latency to respond following a correct (F[3,60]=1.980, p=0.127) or an incorrect response (F[3,60]=1.497, p=0.224).

Organ weights

There was no significant effect of exposure on the size of the uterine horn

(F[3,60]=1.324, p=0.275, Figure 3.6A). None of the doses of ISL altered the size of the uterine horn significantly from those of the OVX vehicle control animals.

There was no significant effect of exposure on the liver to body weight ratio

(F[3,59]=0.963, p=0.416, Figure 3.6B). None of the doses of ISL altered the size of the liver relative to the body weight significantly from those of the OVX vehicle control group.

Discussion

The present study investigated the effects of oral exposure to isoliquiritigenin (ISL) on performance of an operant delayed spatial alternation (DSA) task in middle-aged rats. Daily exposure to 19, 38, or 76 mg/kg body weight of ISL had no measurable effect on DSA performance. Exposure to ISL also did not affect learning in the cued alternation (CA) or non- cued alternation (NCA) training phases prior to DSA testing, and did not affect latency to respond in the DSA task.

Previous work from our research group has shown that treatment with 17β-estradiol tends to impair performance on the DSA task in young (3 month old), middle-aged (12 month old), as well as old (18 month old) OVX rats (Neese et al., 2010a; Wang et al., 2008; Wang et al., 2009).

Additionally, we have shown that genistein, an ERβ selective soy isoflavone impairs performance on the DSA task in middle-aged (14 or 16 month-old) and old (22 month-old) OVX rats (Neese et al., 2010b; Neese et al., 2012). We have also shown that the ERβ agonist DPN impairs performance on the DSA task, but that the ERα agonist PPT does not, suggesting that the 17β-estradiol induced deficit in DSA performance may be ERβ mediated (Neese et al., 2010a).

In the present study, we hypothesized based on these previous studies that ISL, which has been shown to have estrogenic activity both in vitro and in vivo (Maggiolini et al 2002; Miksicek et al, 1993), would also impair performance on this task. LIQ, the active metabolite of ISL, has a twenty-fold higher affinity for ERβ over ERα (Mersereau et al, 2008). Contrary to our hypothesis, we found that ISL at the doses tested had no effect on DSA performance. One possibility is that the levels of ISL in the serum were not high enough to elicit an estrogen-like response. While serum levels of ISL were not measured in this study, based on a pilot study conducted in our lab we estimate that the peak serum levels of aglycone ISL+LIQ in the animals in the present study

55 were between 0.5 and 1 μM. It is useful to look at combined ISL+LIQ levels because both compounds bind to the estrogen receptor and because the two compounds convert back and forth readily in vivo (Simmler et al., 2013). Miksicek et al (1993) found that ISL binds directly to the estrogen receptor in vitro and competes with estradiol for binding. They found that a range of 0.1-

1µM of ISL is needed for a half-maximal response, compared to 0.2nM of 17β-estradiol. While

Miksicek et al. (1993) used an in vitro model and we dosed animals with ISL in vivo, given their results it seems likely that the serum levels of ISL in our animals were high enough to have significant binding affinity at the estrogen receptor. Additionally, while the binding affinities of

ISL and LIQ for the estrogen receptors are substantially lower than that of estradiol, LIQ appears to have greater potency and efficacy through ERβ in gene regulation than expected from its ERβ- binding affinity (Jiang et al., 2013). In an in vitro culture of MCF-7 cells containing ERα or ERβ receptors, a 1μM concentration of LIQ was able to stimulate both PgR and OTUB2 gene expression respectively, indicating that LIQ was able to induce transcriptional activity through both estrogen receptors at that concentration (Jiang et al., 2013). These results suggest that the levels of ISL+LIQ found in the present study were likely high enough to be able to affect transcriptional activity through the estrogen receptors. Additionally, the time course pilot study conducted in our lab indicates that levels of aglycone (bioactive) ISL and LIQ remain elevated in the serum for several hours after oral dosing, indicating that the lack of cognitive effects seen in this study were likely not due to rapid metabolism of ISL or LIQ (Figure 3.1). This is in contrast to an investigation of S-equol done previously in our lab in which the low transcriptional potency of S-equol as well as its rapid metabolism could have led to its lack of cognitive effects (Neese et al., 2014). Importantly, in the present study, ISL was administered orally, thus making the route of exposure relevant to human exposure. In addition, in another recent study we found that

56 treatment with ISL, in the absence of other dietary manipulations, improved performance on a hippocampus sensitive task in OVX rats and the serum ISL+LIQ levels measured in that study were similar to those found in the current study (Kundu et al., 2016). This suggests that the levels of ISL+LIQ in the present study were high enough to have effects in the brain, but that ISL, unlike most other estrogens tested to date, does not impair performance on this operant version of the

DSA task. There is evidence that ISL and LIQ cross the blood brain barrier (Srihari et al, 2012).

However, it is a limitation of the current study that we do not have measurements of these compounds in the relevant brain areas.

It should be noted that while no significant effects of ISL exposure were observed in the current study, there was a trend for the lowest dose ISL group to have the lowest proportion correct across blocks of testing. Additionally, across delays, all exposure groups were below controls with this difference being clearest in the lowest dose group. While these effects were not statistically significant, they are in line with previous research from our group on the ERβ agonist DPN. Neese et al. (2010a) found that while a low dose of DPN impaired performance on the DSA task in OVX middle-aged (12 month old) rats, the two higher doses of DPN did not. Non-monotonic dose curves like that seen in Neese et al. (2010a) are common for compounds that act on hormone receptors

(Vandenberg, 2014). Thus, future studies should expand the dose range to include both lower and higher doses of ISL in order to confirm whether a non-monotonic dose–response curve exists for

ISL on the DSA task.

Organ weights

At the time of sacrifice, uterine horns and livers of all animals were weighed. Animals treated with 19, 38, or 76 mg/kg body weight of ISL had no differences in the size of the uterine

57 horn compared with OVX animals given sucrose pellets only. ISL has the potential to act through estrogen pathways. However, the estrogen-mediated uterotrophic effect is largely an ERα- mediated effect, and since these botanicals are ERβ-selective, the lack of an effect on uterine weight is not surprising and is consistent with the lack of a uterotrophic effect from ISL treatment of adult OVX C57BL6 mice (Malak-Erdogan et al., 2016). Additionally, while ISL and LIQ have some potential to affect ERα-mediated gene expression, in addition to the lower binding affinity for the ERα receptor, these compounds fail to form the stable ER-coregulator complexes that are required for stimulation of reproductive tissues (Malak-Erdogan et al., 2016).

The liver to body weight ratio of animals treated with ISL also did not differ from those of sucrose controls. An increase in liver size is suggestive of increased hepatic enzyme induction, which is a sign of possible toxicity (Maronpot et al, 2010). Licorice root does contain glycyrrhizin, which has been implicated for its hepatotoxic effects at high doses (Isbrucker and Burdock, 2006), however, the pure compound ISL does not. Thus, exposure to ISL did not have any overt clinical effects on uterine proliferation or hepatic toxicity as measured in the current study.

Summary and Conclusions

In the present study we found that daily exposure to 19, 38, or 76 mg/kg body weight of isoliquiritigenin (ISL) had no effect on performance on the prefrontal cortex-sensitive delayed spatial alternation (DSA) task in middle-aged (12 month old) OVX rats. Exposure to ISL also did not alter learning in the cued alternation (CA) or non-cued alternation (NCA) training phases prior to DSA testing. This is in contrast to previous studies from our group showing that exposure to other estrogens, including 17β-estradiol, genistein, and the ERβ agonist DPN, impairs performance on this DSA task in OVX rats. Specifically, previous work from our group suggests that the deficit

58 in DSA performance seen with administration of estrogens may be ERβ mediated. Thus, given that

LIQ, the active metabolite of ISL, has a twenty-fold higher affinity for ERβ over ERα and did not cause a deficit in DSA performance in this study is significant. These results suggest that ISL may not carry the same cognitive risks as most other estrogens tested to date. However, further studies are needed to determine whether there are any negative effects of ISL on other prefrontally mediated tasks or on tasks mediated by other brain regions. In addition, a broader range of ISL doses spanning the dose range that humans could conceivably be exposed to from dietary supplements should be investigated for their biological effects to ensure that there are not negative effects at doses outside the range assessed in this study.

Figures

A B

Figure 3.1. Serum ISL and LIQ levels from a pilot study prior to the current study in female rats (n=6) provided 1 pellet containing 10 mg ISL (dose of ~32 mg/kg body weight. A) Total serum levels of ISL and LIQ across a 24 hour period. B.) Aglycone serum levels of ISL and LIQ across a 24 hour period.

Figure 3.2. There was no main effect of exposure on the days to criterion in the autoshaping phase of training (n=16).

A B

Figure 3.3. A) There was no main effect of exposure on the total number of errors during the cued alternation phase of training (n=16). B) There was no main effect of exposure on the days to reach criterion on the cued alternation phase of training (n=16).

A B

Figure 3.4. A) There was no main effect of exposure on the proportion correct during the non-cued alternation phase of training (n=16). B) All groups increased in their proportion correct across sessions of NCA, but did not differ significantly from each other (n=16).

Figure 3.5. A) (Left panel) There was no main effect of exposure on the proportion correct in the DSA task. There was also no interaction between exposure and block, indicating that all groups performed similarly within testing blocks (n=16). B) (Right panel) There was no interaction between exposure and delay in the DSA task, indicating that all groups performed similarly within each delay (n=16).

Figure 3.6. A) (Left panel) There was no main effect of exposure on the size of the uterine horn (n=16). B) (Right panel) There was no main effect of exposure on the liver weight to body weight ratio (n=16).

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CHAPTER 4: LICORICE ROOT COMPONENTS MIMIC ESTROGENS IN AN OBJECT LOCATION TASK BUT NOT AN OBJECT RECOGNITION TASK

Abstract

This study investigated the efficacy of components of licorice root to alter performance on two different recognition tasks, a hippocampus-sensitive metric change in object location (MCOL) task and a striatum-sensitive double object recognition (DOR) task. Isoliquiritigenin (ISL), licorice root extract (LRE), and whole licorice root powder (LRP) were assessed. Young adult female rats were ovariectomized (OVX) and exposed to ISL, LRE or LRP at 0.075%, 0.5% or 5% respectively in the diet. An estradiol group was included as a positive control based on our prior findings. Rats were allowed to explore two objects for three 5-min study trials (separated by 3-min intervals) before a fourth 5-min test trial where the objects were moved closer together (MCOL task) or replaced with two new objects (DOR task). Rats typically habituate to the objects across the three study trials. An increase in object exploration time in the test trial suggests the rat detected the change. LRP had no effect on recognition while exposure to ISL and LRE improved MCOL performance. Estradiol improved MCOL performance and impaired DOR performance, similar to previously shown effects of estradiol and other estrogens, which tend to improve learning and memory on hippocampus-sensitive tasks and impair striatum-sensitive cognition. Exposure to ISL,

LRE and LRP failed to attenuate DOR, contrary to effects of estradiol shown here and to previous reports in young-adult OVX rats. These findings suggest components of licorice root may prove to be effective therapies targeting memory enhancement without unintended deleterious cognitive effects.

Introduction

Botanical estrogens are non-steroidal plant compounds that can mimic estrogens in the body

(Glazier and Bowman, 2001). These compounds are widely sold as dietary supplements despite a dearth of research on their health effects. Many over the counter botanical supplements contain licorice root powder (LRP) or licorice root extracts (LREs), shown to exert estrogenic effects both in vitro in MCF7 breast cancer cells and in vivo in various tissues including the heart and the pituitary (Maggiolini et al., 2002, Tamir et al., 2001). Isoliquiritigenin (ISL) and liquiritigenin

(LIQ) are two of the primary bioactive compounds in licorice root (Mersereau et al., 2008,

Miksicek et al., 1993). ISL and LIQ convert readily back and forth in the body (Simmler et al.,

2013). Both compounds are lipophilic and have relatively low molecular weight and thus likely cross into the brain through the blood brain barrier (Srihari et al., 2012).

Estrogens have broad ranging actions believed to modulate cognition (for reviews see Galea et al., 2017; Korol and Pisani, 2015; Korol and Wang, 2017; Luine and Frankfurt, 2015).

However, the relationship between estrogens and cognition is a complicated one. The effects of estrogens on cognition vary widely depending on a variety of factors including the cognitive task and the brain areas engaged by the task. Much of the existing literature suggests that estrogen supplementation to ovariectomized (OVX) rodents improves performance on hippocampus- sensitive tasks, but impairs performance on striatum-sensitive tasks. (e.g Davis et al., 2005;

Korol and Kolo, 2002; Pisani et al., 2012). Studies using the same basic training paradigm to assess different cognitive attributes are especially useful in comparing the effects of estrogens on different brain systems, given that several factors like the dose of estradiol used, the pattern of administration as well as aspects of the task environment are held constant. For example,

67 compared to OVX vehicle-treated controls, administration of estradiol to OVX young adult female rats improves performance on an allocentric, place learning version of a 4-arm radial maze task, known to rely on intact functioning of the hippocampus (Chang and Gold, 2003), but impairs performance on an egocentric, response learning version of the same maze task that engages the striatum during learning (Davis et al., 2005; Korol and Kolo, 2002; Korol and

Pisani, 2012; Zurkovsky et al., 2006). Moreover, two days of oral dosing with the estrogen receptor (ER) ERβ-selective botanical estrogen genistein produced a similar shift in learning strategy by enhancing place learning while impairing response learning. These bidirectional effects are not limited to ERβ activation, as several estrogen receptor agonists have also been investigated using this place and response learning paradigm (Korol and Pisani, 2015). The ERα- selective compound propyl pyrazole triol (PPT) and the ERβ-selective compounds diarylpropionitrile (DPN) and Br-ERb-041 were all found to improve place learning and impair response learning, albeit at different doses (Pisani et al., 2015).

There is also some evidence to suggest that supplementation of estrogens to young-adult female rodents impairs performance on prefrontal cortex-dependent tasks. We have used the delayed spatial alternation (DSA) task to investigate working memory in rodents. DSA is thought to primarily engage the prefrontal cortex rather than the hippocampus at delays of less than 15 seconds (Maruki et al., 2001). Using this task, we have found that compared to OVX controls administration of 17-β estradiol impairs performance on the DSA task in young adult OVX rats

(Wang et al., 2008, Wang et al., 2009) as well as in middle-aged and old rats (Neese et al., 2010;

Wang et al., 2009). Similarly, Wide et al. (2004) found that a high dose of estradiol benzoate impairs performance on a non-spatial working memory task at long delays in OVX adult female rats.

Taken together, the findings suggest that estrogen supplementation to OVX young adult rodents facilitates learning and memory that depends on the hippocampus and impairs cognition that depends on the striatum or prefrontal cortex. However, there are exceptions to this pattern of results. When OVX adult rats were tested on four different radial maze tasks that tap the hippocampus, prefrontal cortex, or amygdala, treatment with estradiol benzoate impaired performance on all the tasks except for the prefrontal cortex-hippocampus dependent delayed win-shift task (Galea et al., 2001). Thus, while some studies have found mixed results of estrogen supplementation to rodents, the majority of the literature supports the overarching idea that hippocampal functions are enhanced while non-hippocampal functions are impaired by estrogens and that these bidirectional effects depend on dosing, timing of exposure, and other aspects of the animal history and training environment such as stress history, parity, and age

(Korol and Pisani, 2015).

Estrogens have also been shown to affect performance on object recognition and placement memory in paradigms developed to match task attributes much like the place and response learning paradigm described above. Administration of estrogens to OVX adult rodents enhance performance on hippocampus-sensitive object location tasks (Luine et al., 2003; Tunur et al.,

2012). The effects of estrogens on object recognition are mixed with some evidence that estrogens enhance performance on these tasks (Gresack and Frick, 2006) and some evidence that it impairs performance (Tunur and Korol, 2015). However, the version of the object recognition task most commonly used (e.g Gresack and Frick, 2006) differs from ours (e.g. Tunur and Korol (2015), with some evidence suggesting that the former version is hippocampus-sensitive while the latter version is not (Fernandez et al., 2008; Korol and Pisani, 2015; Tunur and Korol, 2015).

Diet has also been shown to affect cognition. Several studies have shown that a high fat diet can impair various aspects of cognition including spatial learning, working memory, object recognition, and fear conditioning (for a review see Freeman et al., 2014). Impairments have been found on a hippocampus-sensitive radial arm maze task (Granholm et al., 2008; Winocur and

Greenwood, 2005), a prefrontal cortex-dependent operant delayed spatial alternation task

(Winocur and Greenwood, 2005), an operant-based delayed matching to position task (McNeilly et al., 2011), and an object recognition task (Camer et al., 2015; Carey et al., 2014; Jurdak and

Kanarek, 2009; Kaczmarczyk et al., 2013). However, it is worth noting that nearly all of the studies investigating the effects of a high fat diet on cognition in a rodent model, including all of the studies mentioned above, used only male rodents. In the few studies investigating both males and females, some have found sex differences in response to a high fat diet in peripheral metabolism, performance on a contextual fear conditioning task, as well as the magnitude of long-term potentiation (Hwang et al., 2010; Underwood and Thompson, 2016), highlighting the need to investigate the effects of a high fat diet in females as well as males.

In the present studies, we investigated the effects of ISL, LRE and LRP on a hippocampus- sensitive task and a striatum-sensitive task. The metric change in object location (MCOL) task is hippocampus-sensitive (Goodrich-Hunsaker et al., 2008, Korol and Pisani, 2015). Rats habituate to two objects, which are then moved to different spatial locations. The double object recognition

(DOR) task is striatum-sensitive (Korol and Pisani, 2015). Here, rats habituate to two objects, which are then replaced with two new objects. Importantly, the tasks differ only in the final trial and are otherwise identical. Thus, the roles of ISL, LRE and LRP on two distinct cognitive tasks engaging distinct brain areas/memory systems can be compared using these tasks. We chose to investigate LRP and LRE because those are the forms of licorice root typically found in dietary

70 supplements. We included the pure compound, ISL because it is a component of licorice root that has demonstrated estrogenic properties both in vitro and in vivo (Maggiolini et al, 2002; Miksicek et al, 1993; Tamir et al 2001).

To investigate the potential for botanical estrogens to affect cognition in the absence of ovarian hormones, we used an OVX rat model. We wanted to study the effects of ISL, LRE and

LRP in this low-hormone state to investigate the ability of these botanical estrogens to impact cognition in the absence of endogenous estrogens that would compete for ERs. Additionally, we wished to address the issue that the standard rodent diet is much lower in fat than the typical western diet, and previous rodent studies have reported cognitive deficits in rats fed high fat diets relative to those fed standard laboratory chow. Thus, the inclusion of high fat diet groups allowed us to evaluate a diet that more closely models the typical western diet consumed by humans and to investigate whether these botanicals interact with this high fat diet to produce a pattern of effects different from that seen in rats consuming a low fat diet.

Materials and Methods

Animals and treatment

Due to the large number of rats required in these studies, they were each conducted in a series of cohorts or replicates, as described below. The effects of ISL, LRE and LRP on the

MCOL task were investigated in three separate studies consisting of three cohorts each. The effects of ISL, LRE and LRP on the DOR task were investigated in one study consisting of three cohorts. Estradiol groups were also included in all of the studies because previous work has shown that estradiol improves performance on the MCOL task and impairs performance on the

DOR task in OVX young adult rats (Korol and Pisani, 2015; Tunur et al., 2012; Tunur and

Korol, 2015). For each MCOL study, 72 young adult virgin female Long-Evans rats (53 days old) were obtained from Harlan (Indianapolis, IN) in 3 cohorts of 24 rats each, spaced 1 week apart. In each of the three cohorts 4 rats were assigned to each of 6 treatment groups (high fat control, low fat control, high fat estradiol, low fat estradiol, high fat botanical, low fat botanical).

With all cohorts included, there was a total of 12 rats per treatment group in each of the three studies (Table 1A). For the DOR study, a total of 90 female Long-Evans rats was obtained from

Harlan (Indianapolis, IN) in cohorts of 30 rats each, spaced 1 week apart. In each cohort 4 rats were assigned to each of five treatment groups (control, estradiol, ISL, LRE and LRP). With all cohorts included, there was a total of 12 rats per treatment group (Table 1B).

Rats were housed in a temperature and humidity controlled room (22 °C, 40–55% humidity) on a 12-h light–dark cycle (lights on at 7:00 am). Rats were pair-housed in standard plastic cages (17.7× 9.4 × 7.9 inches) with Beta Chip® bedding, and food and water were available ad libitum. The housing facility was fully accredited by the Association for the

Assessment and Accreditation of Laboratory Animal Care (AAALAC). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of

Illinois at Urbana-Champaign and were in accordance with the guidelines of the Public Health

Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (NIH, 1986; Van Sluyters and

Obernier, 2004).

In the MCOL studies the diet was switched from standard rodent chow (Harlan 8604) to either a high fat (TD.06415) or a low fat (TD.94045) diet (Harlan, Madison, WI) on the third day after arrival to the vivarium. Both diets were phytoestrogen free to ensure no additional exposure

72 to estrogenic compounds in the food. The high fat diet included 44.8% kcal from fat with a fatty acid profile as follows: 36% saturated, 47% mono unsaturated, 17% polyunsaturated. The low fat diet included 17.2% kcal from fat with a fatty acid profile as follows: 16% saturated fat, 23% mono unsaturated, 58% polyunsaturated (See table 2). Vitamins and minerals were balanced in the two diets. In the DOR study, the diet was switched from standard rodent chow (Harlan 8604) to the same low fat diet (TD.94045) used in the MCOL studies on the third day after arrival to the vivarium. Given the lack of diet effects in all three of the MCOL studies and limited resources, we did not include high fat groups for the DOR study. Food and water were available to all rats ad libitum. Both rats and their remaining food were weighed every three days so that food intake and the amount of the botanical consumed per µg body weight could be estimated.

Rats with same treatments were pair housed, so intake for each rat was estimated to be half of the total intake for that cage.

In both the MCOL and the DOR studies, all rats were OVX 9 days after arrival. Rats were allowed to recover from OVX for one week before experimental diets were started. ISL,

LRE or LRP was incorporated into the diet of a subset of the animals at a concentration of

.075%, 5% or 0.5% of the diet respectively for three weeks prior to testing. ISL, LRE and LRP were obtained from the Botanical Center at the University of Mississippi. ISL and LRE were purified through methanol extraction. LRP was made by powdering the whole root. The identity and purity of all botanical components was verified by chromatography and spectroscopy. The

ISL was over 95% pure. The concentrations were selected for relevance to predicted human consumption in dietary supplements as detailed in Madak-Erdogan et al. (2016).

Four weeks after OVX and two days before testing, rats in the estradiol groups received a single daily injection of estradiol in sesame oil (s.c. 45µg/kg), 48 and 24 hours prior to testing.

This dose was selected based on previous work showing estradiol-induced improvements in hippocampus-sensitive tasks, and impairments in striatum-sensitive tasks in OVX young adult rats (Korol and Pisani, 2015; Pisani et al., 2012; Tunur et al., 2012; Tunur and Korol, 2015). All rats in the non-estradiol groups in all studies received vehicle injections of sesame oil (s.c

0.45 mL/kg.of oil). Each rat was tested one time during the light portion of the light/dark cycle.

Testing occurred between 8am and 12pm Monday-Saturday (See Figure 4.1 for timeline).

Behavioral testing

Apparatus

The testing apparatus was an opaque, black Plexiglas chamber measuring 28x28 inches, at the base and 21inches tall and surrounded by a black curtain to block peripheral light. The height of the walls of the chamber reduced the possibility of the rat using visual cues outside of the chamber. In both MCOL and DOR studies, two ceramic stimulus objects, 7-8 inches tall were used. Both objects were cookie jars of similar dimensions and were filled with glass marbles to prevent movement of the objects during testing. The objects were chosen to have patterned exterior surfaces that are easy to clean to remove olfactory cues. For the test session in DOR, two novel objects with similar characteristics were substituted. The novel objects were similar in material, texture, and height to the previous objects, but differed in shape and color. A camera was mounted above the chamber to record behavioral testing sessions.

MCOL procedure

Our MCOL task represents a novelty detection paradigm (as described in Goodrich-

Hunsaker et al, 2008). The behavioral testing was conducted in one session with four 5-minute

74 trials consisting of three study sessions (S1, S2, S3) followed by one test session (T; Figure 4.2).

A 3-minute intertrial interval was imposed between each trial, during which rats were returned to their holding cage. Rats were videotaped during the task with a webcam for later scoring.

At the start of each trial, rats were placed in the same location in the chamber. During S1-

S3, the two objects were placed 16 inches apart in the chamber. For T, the objects were moved symmetrically closer together so that they were 12 inches apart (Figure 4.2). Between each trial and between rats, the objects and the chamber (sides and base) were cleaned with a 10% ethanol solution to minimize odor cues. The positions of the individual objects (left or right), the time of day within the testing window, and the tester were counterbalanced across groups. The two testers were blind to the treatment groups of the rats.

DOR procedure

The DOR task differed from the MCOL task only during T. Instead of changing the distance between objects as in MCOL, the objects were replaced with two new objects that differed in size and shape from the original two objects and from each other (Figure 4.2). All other aspects of DOR were as described above for MCOL.

Behavior analysis

In all studies herein, the videos were scored by two people who were blind to the group assignment of the rat. Reliability between scorers was determined with a test set of videos. A correlation coefficient of greater than 95% between coders was required before coding began.

Measures of exploration were positively correlated between testers for both the MCOL and DOR studies (r(47)=0.996, p=<0.001) and r(47)=0.990, p<0.001) respectively). Object exploration

75 time was recorded for each trial using a stopwatch. Exploration was defined as activity directed at the object: including sniffing, whisking, or looking at the object with the nose pointed towards the object. Sitting or climbing on top of the object was not considered object exploration.

ISL and LIQ concentrations

To determine circulating amounts of ISL and LIQ in the blood, tail vein blood samples were collected from each rat two weeks after starting exposure to the botanical diet and one week prior to behavioral testing, as well as at the time of sacrifice. Blood collection was timed so that it fell within the testing window, which was between 8:00 am and 12:00 pm. Rats were euthanized up to one week after the conclusion of behavioral testing, during which trunk blood was collected, again at a time that fell within the behavioral testing window. Rats in the botanical groups received the compounds in their feed until euthanasia. The concentrations of ISL, LRE and LRP in the diets were chosen so as to result in similar blood concentrations of ISL/LIQ in all three exposure groups.

Blood was centrifuged and serum collected and frozen at -20ºC for later analysis at the National

Center for Toxicological Research (NCTR). Levels of total ISL and LIQ were determined using

LC/MS/MS following quantitative hydrolysis of conjugates by β-glucuronidese/arylsulfatase (H. pomatia; Sigma H-1, 2 units/assay) using procedures previously described (Madak-Erdogan et al.,

2016). Levels of aglycone ISL and LIQ were determined without the enzymatic hydrolysis step.

Organ weights

76 hepatotoxicity (Maronpot et al., 2010). As a bioassay for estrogen activity at ERα (Matthiessen,

2013) the uterine horn was removed and proximal fat and vasculature were trimmed off to yield accurate weight (g) of the horn/length of sample (cm) (described in Pisani et al., 2015).

Statistical analysis

For all studies, the primary dependent variable was the total number of seconds spent exploring both objects during a trial. The derived score for statistical analysis (pattern separation index) was calculated as follows (as described in Goodrich-Hunsaker et al, 2008). The number of seconds spent exploring the objects in the test trial was divided by the sum of the number of seconds spent exploring the objects in the third study trial and the test trial. This measure has been used in previous published literature (e.g Goodrich-Hunsaker et al., 2008), however the data were also analyzed using a simple difference score (T-S3). The pattern separation index scores and the organ weights from the MCOL studies were analyzed via two-way ANOVA with treatment and diet as between-subjects factors and with one-way ANOVAs using treatment as the between subjects variable. The pattern separation index scores and organ weights from the

DOR study were analyzed via one-way ANOVA with treatment as a between subjects factor.

The object exploration times for the MCOL and DOR studies were analyzed via a repeated measures ANOVA with trial as a repeated measures factor and treatment as a between subjects factor. When appropriate, Tukey post hoc tests for pairwise comparisons were done. All statistical analyses were done with Systat for Windows Version 13.1

(systatsoftware.com/products/systat/). Significance was set at p <.05.

Results

Behavioral analysis

MCOL studies

In the ISL MCOL study, the ANOVA revealed a significant main effect of exposure on the pattern separation index (F[2,64]=4.265, p=0.018). There was no significant effect of dietary fat and no interaction between dietary fat and exposure (Figure 4.3A). A Tukey post-hoc analysis revealed that the rats exposed to ISL had a significantly higher pattern separation index score than the control group (p=0.014, Figure 4.3B), indicating that ISL rats performed better on the task than did control rats. The estradiol group showed a similar pattern, but there was not a statistically significant difference between the control group and the estradiol group. The exploration times across trials reflects this, showing habituation in all rats from S1 to S3 as expected (F[2,134]=147.049, p<0.001), and an increase in exploration in T in ISL and estradiol treated rats (Figure 4.3C). In the LRE MCOL study, the ANOVA revealed a main effect of exposure on the pattern separation index (F[2,64]=9.763, p<0.000). Similar to the ISL study, there was no significant effect of dietary fat and no interaction between exposure and dietary fat on the pattern separation index (Figure 4.4A). A Tukey post-hoc analysis revealed that both the estradiol group and the LRE group had significantly higher pattern separation index scores than the control group (p=0.001 and p=0.001 respectively, Figure 4.4B), indicating that both the estradiol and LRE rats performed better on the task than did control rats. As with the ISL study, the exploration times across trials reflects this, showing habituation in all rats from S1 to S3 as expected (F[2,134]=114.097, p<0.001), and an increase in exploration during T in LRE and estradiol treated rats but not in control rats (Figure 4.4C). In the LRP MCOL study, the ANOVA revealed a main effect of exposure on the pattern separation index (F[2,65]=4.508, p=0.015).

There was no significant effect of dietary fat and no interaction between dietary fat and exposure on the pattern separation index (Figure 4.5A). A Tukey post-hoc analysis revealed that the rats exposed to estradiol had a significantly higher pattern separation index score than did the control group (p=0.011, Figure 4.5B), indicating that estradiol rats performed better on the task than control rats. However, there was no significant difference between the LRP group and the control group. Again, the exploration times across trials reflects this, showing habituation in all rats from S1 to S3 as expected (F[2,136]=112.406, p<0.001), and a larger increase in exploration during T in estradiol treated rats compared to both other groups (Figure 4.5C). Analysis of the data from each of the three studies using the simple difference of T-S3 as the dependent measure, yielded the same pattern of effects as was observed using PSEP as the dependent measure.

DOR study

The ANOVA revealed a significant main effect of exposure (F[4,54]= 6.591, p<0.001). A

Tukey post-hoc analysis revealed the rats exposed to estradiol had significantly lower pattern separation index scores than all other groups (Figure 4.6). There was no significant difference between the control group and any of the botanical groups. The exploration times across trials reflects this, showing habituation in all rats from S1 to S3 as expected (F[2,106]=157.633, p<0.001), and the smallest increase in exploration during T in estradiol treated rats (Figure 4.6C).

Analysis of the data from each of the three studies using the simple difference of T-S3 as the dependent measure, yielded the same pattern of effects as was observed using PSEP.

Serum ISL and LIQ levels

For the purposes of this analysis, levels of isoliquiritigenin and its active metabolite liquiritigenin were combined because ISL and LIQ readily convert back and forth in the body and both compounds have actions at estrogen receptors (Maggiolini et al., 2002; Mersereau et al.,

2008; Miksicek et al., 1993; , Simmler et al., 2013; Tamir et al., 2001). Analysis of blood samples from the MCOL studies showed that rats treated with ISL, LRE or LRP had significantly elevated levels of ISL+LIQ, with approximately 7%, 2% and 2%, respectively of that being aglycone, which is the active form. The mean serum levels of ISL+LIQ for the ISL,

LRE and LRP groups were 2.71, 6.20 and 8.66 pmol/μl respectively (Figure 4.7A). The mean serum levels of aglycone ISL+LIQ for the ISL, LRE and LRP groups were 0.18, 0.12 and 0.21 pmol/μl respectively (Figure 4.7B). The levels of ISL+LIQ in control rats was lower than the level of detection.

Organ weights

The analysis of uterine horn size in all three of the MCOL studies showed that only estradiol treated animals differed significantly from controls. Because of this, the data were combined across the three MCOL studies for uterine horn size. In the MCOL (combined) and

DOR studies, an ANOVA revealed a significant main effect of exposure on uterine horn size

(F[4,192]=41.871, p<0.0001 and F[4,84]=22.210, p<0.0001 respectively; Figure 4.8). A Tukey post hoc analysis revealed that rats exposed to estradiol had a significantly larger uterine horn than did rats in all other groups (p<0.0001 for all, Figure 4.8). Rats in the botanical groups did not differ significantly from rats in the control group. In the MCOL studies, there was no effect

80 of dietary fat on uterine weight and no interaction between exposure and dietary fat (Figure

4.8A).

In both the ISL and LRE MCOL studies, an ANOVA revealed significant effects of exposure (F[2, 64]=3.210, p=0.047 and F[2,64]=5.978, p=0.004 respectively) as well as dietary fat (F[1,64]=13.858, p<0.001 and F[1,64]=20.990, p<0.001 respectively) on the liver to body weight ratio (data not shown). However, the exposure effect was driven by estradiol treated rats having slightly smaller livers than those in both other groups. ISL and LRE treated rats did not differ from control rats in liver weight. High fat rats had slightly smaller liver to body weight ratios across all groups. In the LRP MCOL study, an ANOVA revealed no significant effect of exposure (F[2,64]=2.364, p=0.102) and no effect of dietary fat (F[1,64]=3.116, p=0.082) on the liver to body weight ratio. In the DOR study, the ANOVA revealed no main effect of exposure on the size of the liver relative to the body weight.

Discussion

The current study investigated the effects of ISL, LRE or LRP on performance on the

MCOL task, which assesses hippocampal function, or the DOR task, which assesses striatal function in young adult, OVX Long-Evans rats. Treatment with ISL or LRE significantly improved performance on the MCOL task compared to OVX control rats. Treatment with estradiol also improved performance relative to control rats with the effect reaching statistical significance in the

LRP MCOL and LRE MCOL studies, but not in the ISL MCOL study. In the DOR study, the estradiol group was impaired on the DOR task compared to the control group and to all three botanical groups. For DOR, none of the botanical groups differed significantly from the control group. However, it should be noted that in all of the studies reported here, the control group was

OVX rats without hormone or botanical supplementation; intact (non-OVX) rats were not included. Thus, it is difficult to know the effects of these compounds in the presence of naturally occurring ovarian steroids.

MCOL task

In the MCOL studies, we found that treatment with ISL or LRE significantly improved the performance of OVX rats on this hippocampus-sensitive task, whereas treatment with LRP did not affect performance on the task. We also analyzed these data using an alternative dependent measure (T-S3) and obtained similar results to the ones reported here. Interestingly in the ISL study, the effect of ISL on task performance was greater than the effect of estradiol. This was an unexpected finding given the relatively low affinity of ISL for the estrogen receptor compared to estradiol. Both estradiol- and ISL-treated rats performed better than control rats did, but the effect was only significant for ISL treated rats. This fits well with our preliminary findings showing that although estradiol supplementation did improve performance on this task, the administration of

DPN, an ERβ agonist, led to a larger improvement in task performance than administration of PPT, an ERα agonist. PPT led to an improvement similar in magnitude to that of estradiol (Tunur et al.,

2012). These findings suggest that ERβ-selective compounds may be more effective than ER-α selective compounds or estradiol in improving performance on this task.

Our hypothesis that treatment with estradiol would significantly improve performance on the MCOL task was partially supported. In all of the MCOL studies, the estradiol group did, as expected, have a higher average pattern separation index than the control group. While this effect was significant in the LRP and LRE studies, it was not significant in the ISL study, although the effect was in the same direction. The results from the MCOL studies are consistent with those from

82 several other studies showing that treating OVX rats with estrogens improves performance on hippocampus-sensitive tasks (e.g. Galea et al., 2017; Gibbs, 2000; Korol and Kolo, 2002; Korol and Pisani, 2015; Korol and Wang, 2017; Luine et al, 1998). Specifically, the results from the

MCOL studies are consistent with the finding that treating OVX rats with the ER agonists PPT or

DPN—agonists selective for ERα or ERβ respectively—or estradiol improves performance on the

MCOL task (Tunur et al., 2012). However, there are exceptions to this pattern of results. For example, some investigators have found that the presence of ovarian hormones can impair acquisition of a spatial version of the Morris water maze. Frye (1995) and later Warren and Juraska

(1997) found that higher levels of ovarian hormones in naturally cycling rats impaired performance on the Morris water maze compared to males or cycling females with lower levels of circulating ovarian hormones. Although, later studies found that cycling females show place learning biases at high hormone stages but response learning biases at low hormone stages in a dual-solution T- maze (Korol et al., 2004; McElroy and Korol, 2005). Thus, there are some mixed results in investigations of ovarian hormone levels on spatial learning and memory in naturally cycling rats.

These results could differ from findings using OVX rats supplemented with exogenous estrogens due to the presence of progesterone along with estrogens in naturally cycling rats (e.g Bimonte-

Nelson et al., 2006).

A high fat and a low fat subgroup were included for each exposure group in the MCOL studies. However, there was no significant effect of diet and no interaction between botanical treatment and diet. This is in contrast to findings that high fat diets cause impairments on a variety of cognitive tasks, including hippocampus-sensitive tasks, prefrontal cortex-sensitive tasks, and object recognition tasks (Camer et al., 2015; Carey et al., 2014; Freeman et al, 2014; Jurdak and

Kanarek, 2009; Kaczmarczyk et al., 2013; Underwood and Thompson, 2016; Winocur and

Greenwood, 2005).

The lack of a high fat diet-induced impairment in the MCOL studies could be due to a number of factors. First, most studies examining the effects of a high fat diet on cognition utilized only males, and we studied OVX females (e.g Freeman et al, 2014; Winocur and Greenwood,

2005). Additionally, many studies investigating the effects of a high fat diet on cognition in rodents used levels of fat that were higher than the ones used in the MCOL studies. Several studies have used diets containing as much as 60% kcal from fat, which is well above the upper limit of human consumption (Buettner et al, 2007; Winocur and Greenwood, 2005). There is some evidence that a Western diet (41% kcal from fat) and a very high fat diet (60% kcal from fat) can affect the brain differently. When administered to mice, both diets led to an increase in body weight, only the very high fat diet impaired performance on a T-maze, increased brain inflammation and reduced brain- derived neurotrophic factor (BDNF) levels (Pistell et al., 2010). BDNF signaling plays a crucial role in hippocampal learning and long-term potentiation (Korol et al., 2013; Yamada and

Nabeshima, 2003). This suggests that the cognitive effects of a high fat diet can differ depending on how much fat the diet contains.

We also measured serum levels of ISL and its active metabolite LIQ. For the purposes of the analysis, the levels of ISL and LIQ were combined, because regardless of which compound is fed the two readily convert back and forth in the body. We found that while levels of total ISL+LIQ differed between the ISL, LRE and LRP groups, with rats in the LRP group showing the highest level of total ISL+LIQ, the total levels of aglycone in the three groups were quite similar. Aglycone levels are more biologically relevant because in this free form, the compounds are able to bind to receptors and cause biological activity such as activating transcriptional changes or second

84 messenger cascades. In the MCOL studies, a significant behavioral effect was seen in rats treated with ISL or LRE, but not with LRP, despite the fact that aglycone levels were similar between the three studies. Licorice root powder is a complex mixture containing hundreds of different compounds, many of which are not found in LRE or in the pure compound ISL. It is possible that

LRP contained compounds that counteracted the behavioral actions of ISL, LIQ or both leading to null effects.

DOR task

In the DOR study we found that treatment with ISL, LRE or LRP had no significant effects on recognition in this striatum-sensitive task in OVX rats, contrary to predictions based on previous results that estrogens, including estradiol, would impair performance on this task (Tunur and Korol, 2015). We did indeed find that estradiol treated rats were impaired on the DOR task compared to all other groups. We also analyzed these data using an alternative dependent measure

(T-S3) and obtained similar results to the ones reported here.

While most estrogenic compounds tested to date, including estradiol, PPT, DPN, and genistein, impair performance on striatum-sensitive tasks, ISL, LRE and LRP did not. It is possible that higher doses than the ones used in this study are needed to see a striatum-sensitive learning impairment. It is also possible that these compounds, unlike other estrogens or selective ER modulators, improve hippocampal function without impairing striatal function. We previously found in a dose-effect study that none of the doses of ISL used had an effect on a prefrontally mediated DSA working memory task, (Kundu et al., 2018), similar to our lack of effects on DOR.

However, estradiol as well as the ERβ-selective agonist DPN, were previously found to impair performance on this DSA task (Neese et al., 2010; Wang et al., 2009). In a pilot study conducted

85 prior to our DSA study we found that aglycone levels of ISL+LIQ in the ISL-treated rats were likely within the range of those found in rats in the DOR study, although the method of dosing did differ between the two studies. In our DSA study, rats were dosed with ISL 60 minutes prior to testing each day. In the DOR study, the botanical compounds were mixed into the diet, which was available ad libitum. Thus, while most studies to date have found that administering estrogens to

OVX rodents results in impairment on striatum-sensitive tasks, the compounds tested in the present study did not, suggesting these botanical compounds in particular might avoid one potential adverse side effect of estrogen supplementation on cognition.

Estrogen effects on object location and object recognition tasks

Our results from the MCOL studies are supported by previous literature on object location tasks showing that administration of estrogens to OVX adult rodents enhances performance on these tasks (Luine et al., 2003; Tunur et al., 2012). However, our results from the DOR study are contrary to findings that estrogens can enhance performance on other types of object recognition tasks (Gresack and Frick, 2006; Luine et al., 2003). This could be because the MCOL and DOR tasks used here differ from most novel object tasks in the literature in several important ways.

Previous novel object studies have been conducted by exposing rodents to 2 objects for one training session and then either replacing or moving one of the objects in the test session.

Exploration time of the novel vs the familiar object is then used as the measure of learning. The

MCOL and DOR tasks differ from those tasks because there are three training sessions instead of one, allowing examination of habituation over study sessions. Additionally, both of the objects are replaced or moved in the final session, perhaps changing the cognitive dimensions of the task that require associations between stimuli. Administration of estradiol, DPN, and PPT, improve

86 performance on the MCOL task while estradiol impairs performance on the DOR task (Tunur et al., 2012; Tunur and Korol, 2015). Previous work has shown that with this paradigm, there is a double dissociation between task and brain area, meaning that hippocampal activation does not seem to be necessary for performing the DOR task and striatum activation does not seem to be necessary to perform the MCOL task (Tunur and Korol, 2015; Korol and Pisani, 2015). In contrast, there is some evidence that the traditional novel object paradigm (e.g Gresack and Frick, 2006;

Luine et al., 2003) is hippocampus-sensitive. It has been found that an intrahippocampal infusion of 17β-estradiol enhances performance on this version of the object recognition task via dorsal hippocampal ERK activation (Fernandez et al., 2008). It is also possible that the timing of treatment is partially responsible for these disparate results. In most studies investigating the effects of estrogens on object recognition tasks, estrogens were administered either a few minutes prior to the training session or immediately after it (e.g Gresack and Frick, 2006; Luine et al., 2003;

Walf et al., 2008). Some studies have administered estrogens up to 4 hours before testing on an object recognition task (e.g Luine et al., 2003; Scharfman et al., 2007). However, in the DOR study, estrogens were administered 48 and 24 hours before training began. Thus, it is possible that estrogens can enhance performance on this task if given close to the time of training, perhaps because of their functions on memory consolidation, but not if given 24 hours prior to training. It is also possible that estrogens can enhance performance on this task through the rapid actions of membrane-associated receptors, but that those actions were not seen in the DOR study due to the timing of administration. These factors could also account for the different findings of the DOR study in the current experiment and other published reports on object recognition

Organ Weights

The uterine proliferation we detected is suggestive of estrogenic activity, especially activity at ERα (Matthiessen et al., 2013). As expected, rats in the ISL, LRE and LRP groups in all studies had uterine horns that did not differ significantly in size from those of control rats. ISL, LRE and

LRP can act through estrogen signaling pathways, however, the estrogen-mediated enlargement of the uterus is largely an ERα effect, and these botanicals are ERβ-selective. This is consistent with the lack of a uterotrophic effect from ISL treatment of adult OVX C57BL6 mice in a previous study (Malak-Erdogan et al., 2016). Additionally, while ISL and LIQ have some potential to affect gene expression through the ERα receptor, in addition to the lower binding affinity for the ERα receptor, these compounds fail to form the stable ER-coregulator complexes that are required for stimulation of reproductive tissues (Malak-Erdogan et al., 2016).

Liver toxicity most likely did not account for the collective results following exposures to

ISL, LRP or LRE. Even though licorice root contains glycyrrhizin, which has been implicated for its hepatotoxic effects at high doses (Isbrucker and Burdock, 2006), ISL, LRE and LRP did not significantly increase the size of the liver relative to body size. In two of the three MCOL studies, estradiol treated rats had a smaller liver to body weight ratio than control rats as well as botanical treated rats. It is not clear why this was the case, as this effect has not been previously reported in the literature to our knowledge. Additionally, in two of the three MCOL studies, rats fed a high fat diet had a smaller liver to body weight ratio than rats fed a low fat diet. This effect was partially, but not wholly driven by a higher body weight in rats fed a high fat diet. Thus, there were some inconsistent effects of estradiol supplementation as well as dietary fat on the liver to body weight ratio, but no effect of the botanical compounds ISL, LRE or LRP.

Summary and Conclusions

In the current studies, treatment with ISL or LRE improved performance of OVX rats on the hippocampus-sensitive MCOL task. Treatment with LRP did not affect performance on the

MCOL task. Treatment with ISL, LRE or LRP did not affect performance on the striatum-sensitive

DOR task. This is in contrast to most estrogenic compounds tested to date, which typically improve hippocampally-based cognitive functions but impair striatally-based learning and memory. Taken together, these results suggest that ISL and LRE could potentially be promising selective estrogen receptor modulators (SERMs) for improving some aspects of cognitive function in a low estrogen state. Importantly, in all of the studies referenced above, the botanical compounds were administered orally, making the results particularly applicable to human health. Dietary supplements containing licorice root and licorice root components are currently taken as oral supplements and thus go through first pass metabolism, as they did in the present set of studies.

However, more studies are needed to show how the doses used in the present studies compare to human exposure from supplements and also to ensure that these or higher doses do not have negative effects such as hepatotoxicity. Additionally, the current studies were done in a surgical menopause model, using OVX young adult rats. Since phytoestrogenic dietary supplements are primarily marketed to post-menopausal women, it will be important to perform similar studies with older OVX rats to determine whether the same pattern of improvement on hippocampus- sensitive tasks and no effect on prefrontal cortex-sensitive and striatum-sensitive tasks are observed across aging.

Tables

Table 4.2: fatty acid profiles of the high fat and the low fat diets used in the MCOL study

High fat diet (44.8% kcal from fat) Low fat diet (17.2% kcal from fat) 36% saturated fat 16% saturated fat 47% mono unsaturated fat 23% mono unsaturated fat 17% polyunsaturated fat 58% polyunsaturated fat

Figures

Figure 4.1. Timeline of the MCOL and DOR studies. Rats were approximately 53 days old upon arrival and approximately 90 days old (89-96 days) when tested.

Figure 4.2. In the MCOL procedure, two objects are placed 16 inches apart for three study sessions. After the final study session (S3) the objects are moved symmetrically closer together for the test session (T). In the DOR procedure, two objects are placed 16 inches apart for three study sessions. After the final study session (S3) the objects are replaced with two new objects for the test session (T). (Figure adapted from Korol and Pisani, 2015.)

Figure 4.3. A). (Top left panel) In the ISL MCOL study, there was a main effect of exposure on the pattern separation index, but no effect of dietary fat and no interaction between dietary fat and exposure. Low fat and high fat groups are represented with light or dark bars respectively. B). (Top right panel) In panel B low fat and high fat groups are combined showing the main effect of exposure. Animals exposed to ISL had a significantly higher pattern separation index score than the control group (p=0.012). C). (Bottom left panel) Raw exploration time of both objects in each trial, with low fat and high fat groups combined.

Figure 4.4. A). (Top left panel) In the LRE MCOL study, there was a main effect of exposure on the pattern separation index, but no effect of dietary fat and no interaction between dietary fat and exposure. Low fat and high fat groups are represented with light or dark bars respectively. B). (Top right panel) In panel B low fat and high fat groups are combined showing the main effect of exposure. Both the estradiol group and the LRE group had significantly higher pattern separation index scores than the control group (Control vs Estradiol, p=0.001, Control vs LRE, p=0.001). C). (Bottom left panel) Raw exploration time of both objects in each trial, with low fat and high fat groups combined.

Figure 4.5. A). (Top left panel) In the LRP MCOL study, there was a main effect of exposure on the pattern separation index, but no effect of dietary fat and no interaction between dietary fat and exposure. Low fat and high fat groups are represented with light or dark bars respectively. B). (Top right panel) In panel B low fat and high fat groups are combined showing the main effect of exposure. Animals exposed to estradiol had a significantly higher pattern separation index score than the control group. The LRP group did not differ significantly from the control group (p=0.01). C). (Bottom left panel) Raw exploration time of both objects in each trial, with low fat and high fat groups combined.

Figure 4.6. A). (Left panel) In the DOR study, the ANOVA revealed a main effect of exposure on the pattern separation index. The animals exposed to estradiol had significantly lower pattern separation index scores than all other groups (p<0.05 for all). There was no significant difference between the control group and any of the botanical groups. B). (Right panel) Raw exploration time of both objects in each trial.

16 0.7 uL uL 14 / Figure/ 7. A). The mean serum levels of isoliquiritigenin plus0.6 liquiritigenin for the ISL, LRE12 and LRP groups in the MCOL study. B). The mean0.5 serum levels of aglycone 10 isoliquiritigenin plus liquiritigenin for the ISL, LRE and0.4 LRP groups in the MCOL 8 study. 0.3 6 4 0.2 Concentration in pmol Concentration in pmol 2 0.1 0 0 total (ISL+LIQ) total (ISL+LIQ) total (ISL+LIQ) aglycone (ISL+LIQ) aglycone (ISL+LIQ) aglycone (ISL+LIQ) ISL LRE LRP ISL LRE LRP Figure 4.7. A). (Left panel) The mean serum levels of isoliquiritigenin plus liquiritigenin for the ISL, LRE and LRP MCOL studies. B). (Right panel) The mean serum levels of aglycone isoliquiritigenin plus liquiritigenin for the ISL, LRE and LRP MCOL studies.

Figure 4.8. Data across the three MCOL studies were combined in panels A). (top left

panel) and B). (top right panel) to illustrate that only the estradiol exposed groups

showed increases in uterine horn weight (p<0.05 for all). Low fat and high fat groups

are represented with light or dark bars respectively in panel A). In panel B), low fat and

high fat groups are combined to show the main effect of exposure. There was no effect

of dietary fat and no interaction between exposure and dietary fat on uterine horn

weight in the MCOL studies. Panel C) (bottom left panel) show that was the same was

true in the DOR study (p<0.05 for all).

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CHAPTER 5: POTENTIAL TARGETS OF LICORICE ROOT COMPONENTS IN THE HIPPOCAMPUS

Introduction

I previously found that oral administration of isoliquiritigenin (ISL), one of the primary estrogenic compounds in licorice root, or a methanol extract of licorice root (LRE) led to improved performance on a hippocampus-sensitive metric change in object location (MCOL) task in ovariectomized (OVX) rats (see Chapter 4). Treatment with licorice root power (LRP) did not affect performance. Previous research has shown that the hippocampus is critical for accurate performance on the MCOL task (Goodrich-Hunsaker et al., 2008, Korol and Pisani, 2015), and that treatment with estradiol improves performance on the MCOL task in OVX rats (Korol and

Pisani, 2015). Given that the MCOL task is both hippocampus-dependent and estrogen-sensitive, the goal of this research was to investigate potential molecular targets of ISL, LRE and LRP that are present in the hippocampus, relevant to learning and memory, and modulated by estrogens. I investigated expression of the following genes using RT-qPCR: Bdnf, Ntkr2, Dlg4, Gria1, Gria2,

Grin1, Grin2B, Esr1 and Esr2.

The justification for choosing these targets is described in detail in Chapter 1. In summary,

I chose brain-derived neurotrophic factor (Bdnf) and its receptor TrkB (Ntkr2) because both are important for learning and memory, including hippocampal-dependent learning and memory

(Huang, 2001; Mizuno et al., 2003). BDNF is involved in the growth and differentiation of new neurons and the formation of long-term potentiation (LTP), among other functions (Huang, 2001).

Deprivation of BDNF or TrkB results in severe learning and memory impairments in mice and rats

(Yamada et al., 2002). I chose post-synaptic density protein 95 (PSD-95, Dlg4) because it is a vital scaffolding protein in the synapse and plays an important role in synaptic plasticity (Sheng 2001,

103

Meyer et al, 2014). PSD-95 is thought to be directly involved in synaptic plasticity by determining the AMPA receptor content in a synapse by acting as a binding slot for AMPA receptors (Qian and Turrigiano, 2011; Nikonenko et al., 2008). PSD-95 is also a postsynaptic marker (Li et al.,

2004). In rats, knockdown of PSD-95 using RNA interference leads to the loss of large patches of the post-synaptic density at excitatory synapses (Chen et al., 2011). GluR1 and GluR2 (Gria1 and

Gria2) are both subunits of the AMPA type ionotropic glutamate receptor. The regulation of these subunits is an important component of LTP (Kauer et al., 2006). In mice with knockin mutations to prevent phosphorylation of the GluR1 receptor, there are deficits in LTP and long-term depression (LTD) in the hippocampus as well as impairments on a rapid learning version of the

Morris water maze (Lee et al., 2003). In GluR2 knockout mice, LTP in the hippocampus as well as exploration in a novel object task is disrupted (Jia et al., 1996). NR1 and NR2B (Grin1 and

Grin2B) are subunits of the ionotropic NMDA type glutamate receptor. The NMDA receptor plays a prominent role in hippocampal LTP as well as in spatial learning and memory (Sun et al., 2010).

In fact, NMDA receptors are the major mediators of LTP in the hippocampus (Spencer et al.,

2008). The NR1 subunit is obligatory in the NMDA receptor. There are four distinct isoforms of the NR2 receptor and the isoforms are expressed differentially in different tissues (Ryan and Grant,

2009). Estradiol treatment to OVX rats increases NMDA receptor specific binding in the hippocampus but decreases this binding in frontal cortex and striatum (Cyr et al., 2001). Knocking out NR1 in the CA1 of mice results in impairment in contextual fear memory as well as taste memory (Li et al., 2007). A conditional knock out of NR2B in CA3 results in severely impaired

LTP in that region (Akashi et al., 2009). I chose ERα and ERβ (Esr1 and Esr2) because ISL, LRE and LRP have activity at these receptors (Boonmuen et al., 2016; Maggiolini et al 2002; Mersereau et al, 2008). Additionally, ISL, LRE or LRP could be modulating the other targets I selected

104 through the ERα and ERβ receptors. I also planned to investigate protein expression of a subset of the targets using Western blotting. I planned to prioritize the targets with altered gene expression from the RT-qPCR analysis for protein analysis.

In contrast to the MCOL results, I found that treatment with ISL, LRE or LRP did not affect performance on the double object recognition (DOR) task in OVX rats, whereas estradiol did decrease performance as expected (see Chapter 4). Thus, I prioritized investigating the effects of

ISL, LRE and LRP on the hippocampi of rats from the MCOL studies, and did not study the brains of the rats from the DOR study.

Methods

At the time of sacrifice, rats were anesthetized by carbon dioxide inhalation then decapitated.

The brains were immediately removed, frozen in liquid nitrogen and stored at -80° C until further use. Hippocampi were isolated as follows: the brains were thawed on ice and both the left and the right hippocampus were quickly removed, frozen in liquid nitrogen and stored at -80° C until further use. Only hippocampi from rats on the low fat (LF) diet were included in the present analysis, due to the lack of any effects of diet alone or any interaction between diet and exposure on the MCOL task. Hippocampi from all 12 rats in the LF ISL group and the LF LRE group were used. Only 8 hippocampi were available for analysis from the LF LRP group. I utilized 20 hippocampi from LF control rats and 20 from LF estradiol rats from the ISL, LRE and LRP MCOL studies. These rats were selected to be balanced across the three studies and across the range of pattern separation scores (low, middle and high) within these groups. Hippocampal tissue was prepared as follows. Frozen hippocampi were placed in a metal bowl. Liquid nitrogen was poured into the bowl and the tissues were ground into powder using a cold pestle. The liquid nitrogen was

105 evaporated off and the powdered tissues were placed into 1.5 ml Eppendorf tubes and stored at -

80°C until further use.

RNA isolation and RT-PCR analysis

Total RNA isolation was performed using an RNA MiniPrep Kit (Sigma-Aldrich, St Louis,

MO, USA). RNA was quantified using a Nano Drop Spectophotometer ND-1000. cDNA synthesis was performed as previously reported in Strakovsky and Pan (2011). Two micrograms of cDNA were synthesized in a 20-μl reaction volume using the High-Capacity cDNA Reverse Transcription

Kit (Applied Biosystems) using random primers and a thermal cycler (Applied Biosystems 2700) with the following program: 25°C for 10 min, 37°C for 120 min, 85°C for 5 sec, and a 4°C hold.

Real-time PCR was performed as previously described in Strakovsky and Pan (2011) and

Strakovsky et al (2013). I used 10 ng of cDNA as the template, SYBR Green PCR Master Mix

(Applied Biosystems), and 5 µM of each forward and reverse primer (Table 1) in the StepOne

Plus Real-Time PCR System (Thermofisher Life Technologies) with the following program: 95°C for 10 min, 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec, 55°C for 1 min, and 95°C for 15 sec, with 40 cycles of steps 2 and 3. A serial dilution was done to create a standard curve for quantification. Following each reaction, a dissociation curve was analyzed. The VectorNTI software (Life Technologies, Grand Island, New York) was used to design all primers for real- time PCR analysis (Table 1). Primers were then analyzed by BLAST and synthesized by IDT

(Coralville, Iowa). The house keeping gene encoding ribosomal protein L7a (L7a) was used to normalize all mRNA data. I investigated expression of the following genes: Bdnf, Ntkr2, Dlg4,

Gria1, Gria2, Grin1, Grin2B, Esr1 and Esr2.

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Western Blotting

Protein extraction and quantification

Protein extraction and quantification was performed as reported in Bandara et al. (2016) for the hippocampi of rats from the MCOL study. One protease inhibitor (PI) tablet was dissolved in 10 mL T-PER. Hippocampal tissue was homogenized in T-PER/PI and then centrifuged at

10,000 x g for 10 minutes, at 4°C. The supernatant was then used in the bicinchoninic acid assay

(BCA) protein assay (Thermo Scientific). The protein assay was performed using the directions for the microplate procedure provided in the instructions of the Pierce BCA Protein Assay Kit. In brief, for each sample, 10μL of protein supernatant was diluted in 15μL of TPER-PI so that the measured protein amounts were within the range of the standard curve. The samples and standards were then loaded into the wells of a microplate and incubated for 30 minutes at 37˚C. The absorbance of the samples at 562 nm was then measured using a Multiskan Ascent microplate reader (Type 354; Thermo Scientific). Protein concentrations were calculated using Ascent

Software (v. 2.6, Revision 3.1, Dec. 2003; Thermo Scientific).

Electrophoresis and membrane transfer

The sample was combined with reducing agent, sample buffer and H2O to obtain 20μL of sample volume. To create the molecular weight ladder, 5µL of marker was used. Samples were denatured by heating at 75˚C for 10 minutes. The protein ladder and protein samples were then loaded into the gel. Running buffer was added and the gels were electrophoresed at 167 volts for

1 hour.

107

To prepare the PVDF membrane for transfer, the membrane was soaked in methanol, water, and then transfer buffer. Filter paper, sponges and the gels were soaked in transfer buffer.

The gel was then placed on filter paper and sandwiched in the transfer module between filter paper and sponges on both sides. Transfer buffer was added to the transfer module and then the module was electrophoresed in a cold room for 1.5 hours.

Blocking and probing

Blocking and probing was done according to the specifications of the antibodies used. The house keeping protein alpha Tubulin was used to normalize densities between gels. The following primary antibodies were used: mouse anti-GluR1 monoclonal (1:500) (Santa Cruz Biotechnology, sc-55509), mouse anti-GluR2 monoclonal (1:500) (Santa Cruz Biotechnology, sc-517265), mouse anti-PSD-95 monoclonal (1:500) (Santa Cruz Biotechnology, sc-32290), rabbit anti-alpha tubulin polyclonal (1:5000) (Abcam, ab4074). The following secondary antibodies were used: mouse IgG kappa binding protein conjugated to horseradish peroxidase (1:1000) (Santa Cruz Biotechnology, sc-516102) and goat anti-rabbit IgG H&L (HRP) (1:5000) (Abcam, ab6721).

Statistical analysis

Gene expression data were normalized by dividing the mRNA content (quantity) of the target gene by the mRNA content of the housekeeping gene, L7a for each sample. Protein expression data were normalized by dividing the relative density of each sample by the relative density of the standard. Densities were calculated using ImageJ software

(https://imagej.nih.gov/ij/). For both gene and protein expression results, normalized values were

108 analyzed via one-way ANOVA with exposure as a between-subjects factor using Systat for

Windows Version 13.1 (systatsoftware.com/products/systat/). Significance was set at p<0.05.

Results

mRNA analysis

A one-way ANOVA with exposure as a between-subjects factor revealed no main effect of exposure on expression of Bdnf, Ntkr2, Dlg4, Gria1, Gria2, Grin1, Grin2b, Esr1, or Esr2

(Figures 5.1-5.9). This was also true when each MCOL study was analyzed separately. While there were no statistically significant differences between groups, expression of some genes was reduced in particular groups relative to controls. Gria1 was reduced in rats treated with estradiol as well as the botanical compounds ISL and LRE relative to controls (Figure 5.4). In addition, expression of

Gria2 and Dlg4 was reduced in rats treated with ISL relative to controls (Figures 5.3 and 5.5).

Thus, I prioritized analyzing protein expression of GluR1, GluR2 and PSD-95.

Protein analysis

A one-way ANOVA with exposure as a between-subjects factor revealed no main effect of exposure on expression of GluR1, GluR2, or PSD-95 (Figures 5.10-5.12). This was also true when each MCOL study was analyzed separately.

Discussion

In the present study, I found that exposure to ISL, LRE, LRP or estradiol did not significantly affect gene expression of Bdnf, Ntkr2, Dlg4, Gria1, Gria2, Grin1, Grin2B, Esr1 or

109

Esr2. I also found that exposure to ISL, LRE, LRP or estradiol did not significantly affect protein expression of GluR1, GluR2, or PSD-95. This was an unexpected finding given that these targets were chosen based on previous literature showing that they are modulated by estrogens (see chapter 5, introduction).

Estrogens can modulate gene and protein expression through two major mechanisms, via direct binding to DNA or via tethering to a DNA-bound transcription factor. In direct binding, the

E2–ER complex binds to an estrogen response element (ERE) sequence and interacts directly with coactivator proteins, resulting in altered transcription (Klinge, 2001). BDNF expression, for example, is thought to be modulated by E2–ER binding to an ERE on the BDNF gene (Scharfman and MacLusky, 2006). In tethering, the E2–ER complex interacts with a DNA-bound transcription factor in a way that stabilizes the DNA binding of that transcription factor, which could then affect recruitment of coactivators to that complex to result in altered transcription (Klinge, 2001).

It is thought that BDNF could be a contributing factor to the fluctuations in hippocampal excitability over the female reproductive cycle (Scharfman et al., 2003). Previous literature suggests that gene and protein expression of BDNF in the hippocampus increase with estradiol supplementation in OVX rats (Gibbs, 1998; Pan et al., 2010; Zhou et al, 2005). The primary receptor for BDNF, TrkB, has also shown sensitivity to modulation by estrogens (Pan et al., 2010;

Spencer et al., 2008). Interestingly, one report also found an increase in gene and protein expression of BDNF and an increase in the gene expression of Ntkr2 in OVX rats given soy phytoestrogens compared to control OVX rats (Pan et al., 2010). Rats given soy phytoestrogens or estradiol also performed better in a spatial Morris water maze task compared to OVX controls in that study, suggesting that soy phytoestrogens can mimic the effects of estradiol on spatial memory as well as in modulating levels of BDNF and TrkB (Pan et al., 2010). Additionally, protein levels

110 of BDNF and pTrkB (the activated form of the TrkB receptor) in the hippocampus have been shown to correspond to circulating levels of ovarian steroids in normally cycling rats (Scharfman et al., 2003; Spencer et al., 2008). Levels of BDNF and pTrkB are highest during the proestrus phase of normally cycling rats, suggesting that levels of BDNF and pTrkB are elevated when estrogen levels are highest (Spencer et al., 2008). Thus, gene and protein expression of BDNF tend to be higher in high estrogen conditions, and lowest in low estrogen conditions. However, in the present study, I saw no effect of estradiol or any of the botanical compounds on gene expression of BDNF or TrkB.

There is also evidence from previous literature that PSD-95 is modulated by estrogens (Li et al., 2004; Waters et al., 2009). PSD-95 increases predictably with the estrogen-mediated increase in spine density in the CA1 region of the hippocampus. Thus protein levels of PSD-95 are often used as a marker of estrogen-induced spine formation (Spencer et al., 2008). Estradiol as well as soy phytoestrogen supplementation to OVX rodents has been shown to increase protein levels of PSD-95 in the hippocampus (Li et al., 2004; Pan et al., 2010; Waters et al., 2009). Dlg4 expression was also shown to be increased in hippocampal cells in vitro with exogenous application of estradiol (Chamniansawat et al., 2012). However, in the present study, I saw no effect of estradiol or any of the botanical compounds on gene or protein expression of PSD-95.

Previous literature has also shown that GluR1 and GluR2, both subunits of the ionotropic

AMPA type glutamate receptor, are modulated by estrogens (Waters et al., 2009). Administration of estradiol, PPT or DPN have been shown to increase protein levels of GluR1 in the hippocampus

(Waters et al., 2009). The authors also found that DPN increased protein expression of GluR2 in hippocampus, but estradiol and PPT did not (Waters et al., 2009). This is contrary to our findings,

111 where I found no effect of estradiol or any of the botanical compounds on gene or protein expression of GluR1 or GluR2.

After OVX, there is a decrease in protein levels of the NR1 and NR2B subunits of the ionotropic NMDA type glutamate receptor (Cyr et al., 2001). There is also a decrease in NMDA receptor binding in the hippocampus (Cyr et al., 2001). This decrease is prevented by administration of 17β-estradiol or the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene (Cyr et al., 2001). However, in the present study, I did not observe any changes in gene expression of Grin1 or Grin2 in the hippocampus in response to estradiol or any of the botanical compounds.

I found no changes in gene expression of Esr1 or Esr2 in response to estradiol or any of the botanical compounds. However, the lack of a change in expression of Esr1 or Esr2 does not rule out the possibility that the botanical compounds acted through these receptors, as increased activity at the estrogen receptors might not lead to a change in gene expression of those receptors.

In summary, my findings are contrary to what the published literature suggests. As discussed above, previous research suggests that in OVX rodents, exposure to estrogens increases the gene and/or protein expression of these targets. One possible reason for the discrepancies between the literature discussed above and the results from the present study is the timing of animal sacrifice. In all of the previous studies discussed above, animals were sacrificed 48 hours or less after the last exposure to estrogens. However, in the present studies, rats were sacrificed up to 7 days after the last exposure to estradiol. If increased expression of these targets in response to estradiol is transient, it is possible that I missed that effect by sacrificing the rats several days after the last exposure to estradiol. However, the rats in the ISL, LRE and LRP groups were exposed to

112 the botanical compounds until the time of sacrifice, so a delay between treatment and sacrifice would not explain the lack of effects in the botanical groups.

Due to limited resources, I only measured protein expression of a subset of the RT-qPCR targets. While the abundance of mRNA is often a good proxy for the abundance of protein

(Ramakrishnan et al, 2009), there are some important considerations. The correlation between mRNA levels and protein levels tends to be lower in perturbed biological systems as opposed to systems in a steady state (for a review see Vogel and Marcotte, 2012). The administration of estrogens to OVX rats could be considered a perturbation. Thus, it is possible that some of the chosen targets would have shown altered protein expression in response to treatment with estradiol or the botanicals, despite a lack of changes in gene expression.

Finally, while I hypothesized that the botanical estrogens would affect gene expression similarly to estradiol, there is very little literature on this topic. One report has found effects of soy phytoestrogens on gene and/or protein expression of BDNF, TrkB and PSD-95 in the hippocampus

(Pan et al., 2010), but much more research is needed. I found significant behavioral effects of ISL and LRE on the MCOL task, but given the lack of changes in gene or protein expression of estrogen-sensitive targets, it is possible that these botanicals are acting through non-estrogen pathways to affect behavior. It is also possible that ISL and LRE were acting in brain regions other than the hippocampus. Future research could address these questions by investigating gene and protein expression of a broader range of targets in the hippocampus as well as in other brain regions.

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Tables

Table 5.1. Primer information.

Gene Species Forward sequence and Reverse sequence and Ensembl ID location location Ntkr2 Rat AAGAACGGCAACCT GGTGGCGGAAATGTCTC ENSRNOT00000 GCGGCACA (+998) CTGGACA (+1073) 042145

Dlg4 Rat CCCATCGCCATCTTC GCTTGCTCCTCTGTGATC ENSRNOT00000 ATCCG (+1989) CGCT (+2065) 068493 Gria1 Rat ATGGTGGGAGAACT CAAGGTTATGGTCAAGG ENSRNOT00000 GGTCTA (+1144) GAG (+1206) 073148 Gria2 Rat AAGGAAAAGACCAG CCCCGACAAGGATGTAG ENSRNOT00000 TGCCC (+2834) AA (+2897) 083361 Bdnf Rat ATGCTCACACAACA TGACCCATGCCAGAAGA ENSRNOG00000 CTGCCCA (+2152) GTGA (+2218) 047466

Esr1 Rat TCGGGAATGGCCTT TCTTGAGCTGCGGGCGA ENSRNOG00000 GTTGC (+28) TT (+97) 019358

Esr2 Rat ATCCCTGGCTTGTG TAGCACCTCCATCCAGC ENSRNOG00000 GAG (+1046) A (+1117) 005343

L7a Rat GAGGCCAAAAAGGT CCTGCCCAATGCCGAAG ENST0000032334 GGTCAATCC (+64) TTCT (+127) 5

114

Figures

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 dfLa(en±SEM) ± (mean Bdnf/L7a 0.1 0 Control Estradiol ISL LRE LRP

Figure 5.1: An ANOVA revealed no main effect of

exposure on expression of Bdnf/L7a.

1 0.98 0.96 0.94 0.92 0.9 0.88 0.86 0.84 tr/7 ma SEM) ± (mean Ntkr2/L7a 0.82 0.8 Control Estradiol ISL LRE LRP

Figure 5.2: An ANOVA revealed no main effect of exposure on expression of Ntkr2/L7a.

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 l4La(en±SEM) ± (mean Dlg4/L7a 0.1 0 Control Estradiol ISL LRE LRP

Figure 5.3: An ANOVA revealed no main effect of exposure on expression of Dlg4/L7a.

115

4.5 4 3.5 3 2.5 2 1.5 1 ra/7 ma SEM) ± (mean Gria1/L7a 0.5 0 Control Estradiol ISL LRE LRP

Figure 5.4: An ANOVA revealed no main effect of exposure on expression of Gria1/L7a.

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

li2La(en±SEM) ± (mean Glria2/L7a 0.1 0 Control Estradiol ISL LRE LRP

Figure 5.5: An ANOVA revealed no main effect of

exposure on expression of Gria2/L7a.

1.2

0.8

0.6

0.4

0.2 rn/7 ma SEM) ± (mean Grin1/L7a

0 Control Estradiol ISL LRE LRP

Figure 5.6: An ANOVA revealed no main effect of exposure on expression of Grin1/L7a.

116

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1

rnbLa(en±SEM) ± (mean Grin2b/L7a 0.05 0 Control Estradiol ISL LRE LRP

Figure 5.7: An ANOVA revealed no main effect of

exposure on expression of Grin2b/L7a.

1.6 1.4 1.2 1 0.8 0.6 0.4

s1La(en±SEM) ± (mean Esr1/L7a 0.2 0 Control Estradiol ISL LRE LRP

Figure 5.8: An ANOVA revealed no main effect of exposure on expression of Esr1/L7a.

1.6 1.4 1.2 1 0.8 0.6 0.4

s2La(en±SEM) ± (mean Esr2/L7a 0.2 0 Control Estradiol ISL LRE LRP

Figure 5.9: An ANOVA revealed no main effect of exposure on expression of Esr2/L7a.

117

0.98

0.96

0.94

0.92

0.9

0.88 lR/lh uui ma SEM) GluR1/alpha tubulin(mean ± 0.86 Control Estradiol ISL LRE LRP

Figure 5.10: An ANOVA revealed no main effect of exposure on levels of Glur1/alpha tubulin.

0.98 0.97 0.96 0.95 0.94 0.93 0.92 0.91 0.9

lR/lh uui ma SEM) GluR2/alpha tubulin(mean ± 0.89 0.88 Control Estradiol ISL LRE LRP

Figure 5.11: An ANOVA revealed no main effect of exposure on levels of Glur2/alpha tubulin.

0.9 0.88 0.86 0.84 0.82

SEM) ± 0.8 0.78 0.76 0.74 PSD-95/alpha tubulin (mean (mean tubulin PSD-95/alpha 0.72 Control Estradiol ISL LRE LRP

Figure 5.12: An ANOVA revealed no main effect of exposure on levels of PSD-95/alpha tubulin.

118

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CHAPTER 6: CONCLUSIONS

Estrogens can affect cognition, as reviewed in chapter 1. Botanical estrogens, through their ability to mimic estrogens, also have the potential to affect cognition. Many botanical estrogens are widely available over the counter in dietary supplements, despite a dearth of research about their effects. This work investigated the cognitive effects of components of licorice root, one of the most common sources of botanical estrogens in dietary supplements. Initially, I explored whether the pure compound isoliquiritigenin (ISL) impacted performance on a delayed spatial alternation (DSA) working memory task in ovariectomized (OVX) middle-aged rats. Previous work has found that estrogens impair performance on the DSA task in OVX rats (Neese et al.,

2010; Wang et al., 2008; Wang et al., 2009). In later studies, I expanded to include an extract of licorice root (LRE) as well as whole licorice root powder (LRP). In those studies, object location memory and object recognition memory were assessed in OVX young-adult rats. Young-adult rats were used as a starting point due to the fact that we had never used these tasks in our lab before and previous studies using these tasks to investigate the impact of estrogens were all done in young-adult rats (Korol and Pisani, 2015).

The main objectives of this dissertation were to: 1) determine whether ISL affects performance on an operant working memory task in OVX rats; 2) determine whether ISL, LRE or

LRP affect performance on an object location and an object recognition task in OVX rats; 3) determine whether a high fat diet interacts with the effects of ISL, LRE or LRP on performance in an object location task; and 4) investigate the mechanisms by which ISL and LRE affected performance on the object location task.

In the study described in chapter 3, ISL had no effects on performance on a delayed spatial alternation (DSA) working memory task in middle-aged OVX rats at any of the three doses (6, 12

123 or 24 mg) that were tested. Estradiol has been shown to impair performance on this task in young- adult as well as middle-aged and old OVX rats (Neese et al., 2010; Wang et al., 2008; Wang et al.,

2009). Additionally, the ERβ agonist DPN, but not the ERα agonist PPT, impaired performance on the DSA task, suggesting that the estradiol-induced deficit in DSA performance may be ERβ mediated (Neese et al., 2010). ISL, as well as its active metabolite liquiritigenin (LIQ), have known estrogenic activity and are ERβ selective (Boonmuen et al., 2016; Mersereau et al., 2008, Miksicek et al., 1993). Thus, I hypothesized that ISL would also impair performance on the DSA task. Based on the results of a previous pilot study conducted in our lab, the peak serum levels of aglycone

(bioactive form) ISL+LIQ in the rats in the DSA study were likely between 0.5 and 1 μM. ISL binds directly to the estrogen receptor in vitro and competes with estradiol for binding, with a range of 0.1-1µM of ISL needed for a half-maximal response (Miksicek et al., 1993). Given this, it seems likely that the serum levels of ISL in rats in the DSA study were high enough to have significant binding affinity at the estrogen receptors. Additionally, our pilot study indicated that levels of aglycone ISL and LIQ remain elevated in the serum for several hours after oral dosing, indicating that the lack of cognitive effects seen in the DSA study were likely not due to rapid metabolism of ISL or LIQ. Thus, it was found that unlike most other estrogens tested to date, ISL does not impair performance on this operant DSA task. It is estimated that human exposure to ISL through dietary supplements is approximately 1-2 mg/kg of body weight per day (Madak‐Erdogan et al., 2016). Using allometric scaling, the rat equivalent of that dose range is approximately 4-8 mg/kg per day (Madak‐Erdogan et al., 2016). The doses of ISL in the DSA study were similar to the estimated human exposure (approximately 19, 38, or 76 mg/kg). It is possible that ISL has non- monotonic effects and could affect DSA performance at lower doses. It is also possible that ISL would have different effects on DSA performance if given as part of a complex mixture like LRE

124 or LRP, making the exposure more similar to human exposure through supplements. Thus, more research is needed to investigate those possibilities.

In the studies described in chapter 4, I expanded my goals to look at the pure compound

ISL, as well as LRE and LRP. I also included a diet component to investigate how a high fat diet might interact with exposure to the botanicals. ISL and LRE, like estradiol, improved performance on a hippocampus-dependent metric change in object location (MCOL) task in young-adult OVX rats. LRP had no effect on performance on the MCOL task. There was also no effect of a high fat diet on this task and no interaction between exposure to botanicals or estradiol and diet. Previous research found that estradiol improved performance on the MCOL task in young-adult OVX rats

(Korol and Pisani, 2015; Tunur et al., 2012; Tunur and Korol, 2015), an effect that was replicated in the MCOL studies. Additionally, earlier findings have shown that administration of the ERβ- selective agonist DPN led to a larger improvement in task performance than administration of PPT, while PPT led to an improvement similar in magnitude to that of estradiol (Tunur et al., 2012).

These findings suggest that ERβ-selective compounds may be more effective than ERα selective compounds or estradiol in improving performance on the MCOL task. Although, a direct comparison of the same doses of the agonists might not be appropriate due to the different efficacies of DPN and PPT, the finding that ERβ-selective components of licorice root improved performance on the MCOL task in OVX rats is consistent with previous literature. LRP failed to affect performance on the MCOL task, despite serum levels of aglycone (bioactive form) ISL+LIQ being similar to those in the ISL and LRE treated groups. It is possible that the complex mixture

LRP contained antagonist compounds that blocked the actions of ISL or LIQ. Indeed, a recent analysis of several licorice root components found that some compounds in licorice root bind estrogen receptors, but do not activate transcription, effectively acting as antagonists (Boonmuen

125 et al., 2016). Thus, the complex mixture would not necessarily yield the same effects as the pure compound or the methanol extract. No effect of a high fat diet was found in the MCOL studies.

This is contrary to previous literature showing that high fat diets can impair various aspects of cognition in rodents, including hippocampus-sensitive tasks (e.g Freeman et al., 2014). The lack of a high fat diet-induced impairment in the MCOL studies could be due to a number of factors.

First, most studies examining the effects of a high fat diet on cognition utilized only males, and the present studies utilized OVX females. Additionally, many studies investigating the effects of a high fat diet on cognition in rodents have used diets containing as much as 60% kcal from fat, which is well above the upper limit of human consumption and higher than the 45% kcal from fat used in the MCOL studies (Buettner et al., 2007; Winocur and Greenwood, 2005). It is also possible that the five weeks of exposure to a high fat diet in the MCOL studies was not sufficient to affect performance on this task. Given the lack of effects and large number of animals needed to test both diets, a high fat diet was not tested in the DOR study conducted next.

ISL, LRE and LRP had no effect on a striatum-dependent double object recognition

(DOR) task in young-adult OVX rats. Estradiol was previously shown to impair performance on this task in young-adult OVX rats (Korol and Pisani, 2015; Tunur and Korol, 2015), an effect that was replicated in the DOR study. Estrogens, including estradiol, DPN, PPT and genistein have been shown to impair performance on striatum-sensitive tasks in young-adult OVX rats

(Korol and Kolo, 2002; Korol and Pisani, 2015; Pisani et al., 2012, Pisani et al., 2016). However, unlike most estrogens tested to date, ISL, LRE and LRP did not impair performance on this striatum-sensitive task. The exposure paradigm and doses in the DOR study were identical to those in the MCOL studies where licorice root components modulated performance on an object location task. This suggests that the lack of effects seen with botanical exposure in the DOR

126 study was not due to insufficient exposure to ISL, LRE or LRP. However, it is possible that the dose range needed to see an effect in the striatum is different from that needed in the hippocampus. Thus, I found that ISL and LRE mimicked estradiol by enhancing performance on the MCOL task in OVX-young adult rats, but avoided the impairing effect of estradiol on the

DOR task at the doses tested.

Chapter 5 addressed the goal of investigating potential mechanisms of action for the observed behavioral effects in the MCOL study. The expression of the following genes was measured in the hippocampi of animals from the MCOL study using RT-qPCR: Brain-derived neurotrophic factor (Bdnf), the primary receptor for BDNF (Ntkr2), post-synaptic density protein

95 (Dlg4), subunits of the ionotropic AMPA type glutamate receptor (Gria1 and Gria2), subunits of the ionotropic NMDA type glutamate receptor (Grin1 and Grin2B), as well as the estrogen receptors ERα and ERβ (Esr1 and Esr2). Previous work has shown that these targets are involved in cognition in the hippocampus and are modulated by estrogens, as detailed in chapter 5. There were no significant differences in the expression of any of the selected genes in response to ISL,

LRE, LRP or estradiol. However, there was a trend for reduced expression of Dlg4, Gria1, and

Gria2 in response to botanical or estradiol exposure. Thus, western blotting was used to investigate the protein products of those genes (PSD-95, GluR1 and GluR2 respectively). There were no significant differences in expression of any of those proteins in response to ISL, LRE, LRP or estradiol. These findings were unexpected because previous literature has shown that estradiol increases gene or protein expression of all of the chosen targets in OVX rodents. The lack of changes in gene expression in estradiol exposed animals could be due to the delay between exposure to estradiol and sacrifice of the rats in the MCOL studies. Whereas most previous studies sacrificed animals 48 hours or less after the last exposure to estrogens, rats were sacrificed up to 7

127 days after the last exposure to estradiol in the MCOL studies. If increased expression of these targets in response to estradiol is transient, it is possible that by sacrificing the rats several days after the last exposure to estradiol, that transient increase was missed. However, the rats in the ISL,

LRE and LRP groups were exposed to the botanical compounds in their diet until the time of sacrifice, so a delay between treatment and sacrifice would not explain the lack of effects in the botanical groups. Thus, it is possible that the botanicals acted through non-estrogenic pathways.

For example, ISL has also been shown to act as a vasorelaxant, an antioxidant, an anti- inflammatory agent and an anti-tumor agent (Zhan et al., 2006). It has also been shown to modulate

GABA(B) receptors to inhibit the release of dopamine (Jang et al., 2008), and to be a sirtuin- activating compound (Alcaín and Villalba, 2009).

Importantly, in all the above studies, licorice root components did not significantly affect the size of the liver or uterine horn. An increase in liver size is suggestive of hepatotoxicity

(Maronpot et al, 2010). Uterine proliferation is suggestive of estrogenic activity, especially activity at estrogen receptor (ER)α (Matthiessen et al., 2013). Both of these are undesirable side effects.

It would be useful to conduct additional studies to further evaluate the potential for ISL and LRE to improve cognition in a low estrogen state, such as in post-menopausal women. In the present studies, ISL and LRE mimicked the beneficial effects of estradiol in the MCOL task, but avoided the negative effects of estradiol in the DSA and DOR tasks at the doses tested in these studies. ISL and LRE also do not appear to cause hepatotoxicity or uterine proliferation at the doses that were tested. Future research should investigate the effects of these botanicals in older rats to investigate whether the cognitive effects are similar in an aged brain, given that dietary supplements containing botanical estrogens are marketed primarily to peri- and post-menopausal women. Additionally, a wider range of doses should be investigated. Future work should

128 investigate the range of exposure to licorice root components in humans to ensure that doses used in preclinical models are relevant for evaluating the safety and efficacy of these compounds in human populations. A range of other cognitive tasks should also be investigated to gain a fuller understanding of the effects of licorice root components on cognition. More targets in the brain should also be investigated to uncover the mechanisms of action of these compounds. Other estrogen-sensitive pathways could be investigated in future research, such as the effects of licorice root components on ERK signaling, a pathway known to play a critical role in learning as well as estrogen signaling (Fan et al., 2010; Samuels et al., 2008). Non-estrogenic pathways that have been shown to be affected by ISL that could affect cognition should also be investigated, such as effects on dopamine release, GABA modulation, sirtuin activation, and vasodilation (Alcaín and Villalba,

2009; Jang et al, 2008; Zhan et al., 2006).

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