Reducing luteinizing hormone levels after ovariectomy improves spatial memory: Possible role of brain-derived neurotrophic factor

Hormones and Behavior 118 (2020) 104590

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Reducing luteinizing hormone levels after ovariectomy improves spatial memory: Possible role of brain-derived neurotrophic factor

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Nathaniel Bohm-Levinea, Alexander R. Goldberga, Monica Mariania, Maya Frankfurtb, ⁎ Janice Thorntona, a b

Department of Neuroscience, Oberlin College, 119 Woodland St, Oberlin, OH 44074, USA Department of Science Education, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549, USA

ARTICLE INFO

ABSTRACT

Keywords: Alzheimer's disease Estrogen Luteinizing hormone Brain-derived neurotrophic factor Spatial memory

Alzheimer's disease and other forms of cognitive decline are significantly more prevalent in post-menopausal women. Decreased estrogen levels, due to menopause or ovariectomy, may contribute to memory impairments and neurodegeneration. Another result of decreased estrogen levels is elevated luteinizing hormone (LH). Elevated LH after menopause/ovariectomy has been shown to impair cognition in both human and animal studies. Lowering LH levels rescues spatial memory in ovariectomized (ovx) rodents, yet the mechanisms of these effects are still unclear. Estrogens appear to exert some of their effects on memory by increasing levels of brainderived neurotrophic factor (BDNF) in the hippocampus. In these studies, we explored whether lowering LH may act by increasing BDNF. Ovx rats were treated with Antide, a gonadotropin releasing hormone receptor antagonist that lowers LH levels, or with estradiol. Both Antide and estradiol treatment enhanced spatial memory in ovx females. Both were found to be ineffective when a BDNF receptor antagonist was administered. Immunohistochemical analysis revealed that both Antide and estradiol increased BDNF expression in the hippocampus. Dendritic spine density on pyramidal cells in CA1 was unchanged by any treatment. These results provide evidence for a relationship between LH and BDNF in the hippocampus and demonstrate that estrogenincreasing and LH-lowering treatments may both require BDNF signaling in order to improve spatial memory.

1. Introduction Cognitive decline is a pervasive and growing health issue in both men and women worldwide. The World Health Organization puts the number of individuals suffering from dementia at approximately 47.5 million (World Health Organization, 2015). Alzheimer's disease (AD), the most prevalent form of dementia, is estimated to afflict more than a third of those over 85 (Hebert et al., 2013). Of the 5 million Americans suffering from AD, two-thirds are women (Koran et al., 2017). Apart from the higher prevalence, there are many sex differences in AD, both in terms of symptoms and neuropathology (Koran et al., 2017), which has led researchers to investigate the role of ovarian hormones. Menopause in women is accompanied by a decrease in estrogen production by the ovaries, leading to a reduction of estrogen levels throughout the brain and body (Coffey et al., 1998). Estrogens play a significant role in cognition and cognitive decline. Ovariectomized (ovx) rats have decreased levels of estradiol (the primary estrogen) and exhibit deficits in spatial memory tasks, including the radial arm maze, Y maze, and Barnes maze (Berry et al., 2008; ⁎

Bimonte and Denenberg, 1999; Luine et al., 2003). Generally, female rats display improved performance after implantation with subcutaneous estradiol capsules or estradiol injections (Fader et al., 1998; Luine et al., 1998), although this depends upon estradiol dose and timing (e.g. Daniel and Bohacek, 2010; Frick, 2009). Increasing the period of time between ovx and treatment renders estradiol ineffective at improving cognition and rescuing losses in spine density that may underlie cognitive impairments (Daniel et al., 2006). Studies have shown that a drop in estradiol levels make neurons more vulnerable to degeneration, leading many to view estradiol as neuroprotective (Dubal et al., 1999; Garcia-Segura et al., 2001). Consistent with this theory, women who undergo menopause later in life have a lower risk of developing AD (Henderson, 1994) and surgical removal of the ovaries in pre-menopausal women significantly increases the risk of cognitive impairment (Rocca et al., 2007). In addition to direct effects in the CNS, estradiol controls levels of luteinizing hormone, another hormone in the hypothalamic-pituitarygonadal (HPG) axis. Luteinizing hormone (LH) binds receptors in the ovaries to stimulate production of estradiol and other steroid hormones

Corresponding author. E-mail address: [email protected] (J. Thornton).

https://doi.org/10.1016/j.yhbeh.2019.104590 Received 13 May 2019; Received in revised form 25 August 2019; Accepted 13 September 2019 0018-506X/ © 2019 Elsevier Inc. All rights reserved.

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(Davis, 1994). Estradiol exerts negative feedback by binding receptors in the hypothalamus and pituitary, which in turn leads to decreased LH production and release (Goodman and Lehman, 2012). Consistent with these negative feedback effects, ovx rats have low estradiol levels and elevated LH levels in the blood serum (Wallace et al., 2006). LH is able to cross the blood-brain barrier (Knowles, 1972; Lukacs et al., 1995) and LH receptors are found in higher concentration in the hippocampus than other brain areas (Lei et al., 1993). Multiple studies have shown that high levels of LH negatively impact spatial memory (see Burnham and Thornton, 2015 for review). Transgenic mice that overexpress the LH beta-subunit have increased serum LH levels and decreased performance on spatial memory tasks (Casadesus et al., 2007). Both acute and chronic treatment with human chorionic gonadotropin (hCG), an LH homologue with a longer half-life (Esbenshade et al., 1986), leads to spatial memory deficits in ovx rats even when they have estradiol implants (Berry et al., 2008). Direct infusion of hCG into the dorsal hippocampus also causes spatial memory deficits in estradiol-implanted ovx rats (Burnham et al., 2017). Several studies have observed rescued memory after lowering LH levels. Treatment with Antide, a GnRH receptor antagonist, lowers LH levels back to gonadally-intact levels and improves performance on spatial memory tasks in ovx rats (Ziegler and Thornton, 2010). Leuprolide acetate, a GnRH “super-agonist” that disrupts the HPG axis and lowers LH levels, also rescued spatial memory in a transgenic mouse model of AD (Casadesus et al., 2006). Both Antide and leuprolide acetate were found to be more effective than estradiol in rescuing memory deficits (Palm et al., 2014; Ziegler and Thornton, 2010). Most recently, Burnham et al. (2017) showed that direct infusion of deglycosylated hCG, an LH receptor antagonist, into the dorsal hippocampus rescued spatial memory in ovx rats. While the aforementioned drugs act through disparate mechanisms, their parallel effects on behavior indicate that their shared effect of lowering LH is what underlies behavioral changes. Although the mechanisms by which hormones affect memory are not well understood, there is evidence to suggest that brain-derived neurotrophic factor (BDNF) plays a key role. BDNF is a neurotrophin involved in synaptic plasticity and multiple forms of memory (see review by Luine and Frankfurt, 2013). BDNF acts primarily at the receptor tropomyosin receptor kinase B (TrkB) and activates several signaling cascades, including those important for dendritic spine maintenance (Vigers et al., 2012), and spine/synapse density in cortical and hippocampal neurons (Cohen-Cory et al., 2010). BDNF binding induces TrkB phosphorylation, which was found to be necessary for synaptic plasticity and long-term spatial memory (Lai et al., 2012). Studies of post-mortem tissue have found decreased transcription levels of BDNF and its precursor pro-BDNF in the parietal cortex of patients suffering from AD and mild cognitive impairment (Holsinger et al., 2000). Serum BDNF levels also appear to decrease in patients with AD and vascular dementia (Yasutake et al., 2006). Estradiol regulates both BDNF mRNA and protein expression in a number of brain areas including parts of the cortex, hippocampus and olfactory bulb (Bimonte-Nelson et al., 2004; Miranda et al., 1993; Scharfman et al., 2003; Sohrabji et al., 1995). Estradiol stimulates BDNF mRNA and protein expression but there are regional differences in estradiol's induction of BDNF (Gibbs, 1999; Singh et al., 1995; Sohrabji et al., 1995; Spencer et al., 2008). Ovx rats treated with estradiol have increased levels of TrkB mRNA in the hippocampus (Pan et al., 2010) and gonadally-intact mice express increased levels of TrkB protein during their proestrous phase (Spencer et al., 2008). Estradiol's effects on TrkB are abolished in an estrogen receptor knockout mouse model (Spencer-Segal et al., 2012). Compared to estrogens, much less is known about the relationship between LH and BDNF. While LH appears to increase BDNF levels in gonadal tissue (Feng et al., 2003; Russo et al., 2012; Zhao et al., 2011), a small amount of data suggests that LH actually decreases BDNF levels in the CNS. Palm et al. (2014) demonstrated that reductions in LH due

to leuprolide acetate treatment resulted in increased synthesis of BDNF mRNA in the frontal cortex. In line with these findings, Blair et al. (2016) found increases in cortical spine density in leuprolide-treated rats, a sign of BDNF involvement. It is important to note that these two studies were conducted in cortical tissue, and not in the hippocampus. Further, no studies to date have examined the functional implications of blocking BDNF-TrkB signaling in vivo on spatial memory in rodents. We set out to accomplish the following with the present studies: (1) to determine whether knocking out BDNF-TrkB signaling with the TrkB antagonist ANA-12 would ablate spatial memory improvements in ovx rats treated with estradiol or GnRH-antagonist Antide (which lowers LH levels); (2) to determine whether Antide influences expression levels of BDNF and its activated receptor, phosphorylated TrkB in the hippocampus; and (3) given that estradiol increases dendritic spine density in some brain regions and this has been correlated with improved memory, we assessed the effects of Antide and estradiol on spine density in CA1. 2. Materials and methods 2.1. Animals Adult (4–6 mo old) female Sprague-Dawley rats bred from animals purchased from Hilltop Animal Laboratories, Inc. (Scottsdale, PA) were used in all experiments. Rats were housed in groups of 2–3 in plastic cages that measured 27.9 × 20.3 × 17.8 cm and were kept on 14 h:10 h dark-light cycle at 22 °C. Rats had ad libitum access to water and Purina Rat Chow. All procedures were approved by the Oberlin College Institutional Animal Care and Use Committee. 2.2. Hormones and drugs All animals were ovariectomized (ovx) under continuous isoflurane anesthesia (0.75 l/min O2; 2–3% anesthetic) and a silastic capsule containing estradiol (or left blank) was implanted subcutaneously (s.c.) between the shoulder blades. Estradiol (E) capsules were constructed of 15 mm Silastic tubing (1.57 mm i.d., 3.18 mm o.d.; Dow Corning), and filled to 5 mm in length with crystalline 17β-estradiol (Sigma). Capsules were sealed on both ends with a wood dowel and silicone glue and equilibrated in 0.9% saline for 24 h prior to implantation. Capsules provided constant, circulating levels of estradiol (25–50 pg/ml) based on previous studies (Legan et al., 1975; Luine and Rodriguez, 1994; Thornton, unpublished results). Control capsules (Blank) were prepared similarly but did not contain any hormone. Females were allowed to recover for 4 days after surgery. ANA-12, a low molecular weight, TrkB-specific antagonist (SigmaAldrich; see Cazorla et al., 2011), was prepared in 1% DMSO in 0.9% NaCl saline and delivered i.p. at a concentration of 0.5 mg/ml/kg bodyweight. This dose was determined to be effective in a previous study using ANA-12 in a rat model of cognitive decline (Kishi et al., 2012). GnRH receptor antagonist Antide (Bachem, CA) was dissolved in sterilized MilliQ water and delivered s.c. at a concentration of 1.0 mg/ kg bodyweight. This dose was chosen based on findings by Ziegler and Thornton (2010) demonstrating that LH levels in ovx rats significantly decreased 4 h after injection and stayed low for at least 96 h. All drugs were stored at −20C, made up on the morning of the first test, and subsequently kept at 4C. 2.3. Behavioral assays All behavioral assays were performed in an 80 cm × 80 cm × 30 cm open field arena, marked by a 10 cm × 10 cm grid. External visual cues, including a 55.8 cm × 69.8 cm white cross on a black background on one wall, remained stationary throughout habituation and behavioral testing. For all Object Location Tests and some habituation tests (see below) the floor was covered with wood shavings. Between tests, the 2

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arena and any objects used were cleaned with 70% ethanol, and wood shavings were swirled to disrupt olfactory cues. All behavioral testing took place under red light, at least 45 min into dark cycle.

freeware (NIH). Proteins were normalized to β-actin and quantified relative to the Blank control band for each blot.

2.3.1. Open field habituation and locomotor activity tests Procedures were adapted from Berry et al. (2008). Ovx females were habituated to the test arena over a four-day period. On the first day of habituation, animals were habituated to the arena with wood shavings in groups of 4–6 for 20 min. On the second day, animals were habituated to the arena in the absence of wood shavings, for 20 min in groups of 4–6. On the third day, rats were habituated to the shavingfilled arena individually for 5 min. Animals were handled daily prior to and during the habituation period. Locomotor activity was assayed on the fourth day of habituation, by placing rats individually in the shaving-free open field arena and measuring the number of 10-cm-spaced lines crossed over a period of 5 min. Locomotor activity was also assayed 4–6 h after treatment, following the last spatial memory test.

2.5. BDNF immunohistochemistry Females were anesthetized with pentobarbital and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer and brains were post-fixed overnight and stored in 30% sucrose in PBS. Brains were cut using a freezing microtome into 50 μm coronal sections through the hippocampal area between 2.64 mm and 4.56 mm posterior to bregma (Paxinos and Watson, 2006). Sections were stored in cryoprotectant until used. Sections were treated with 1% H2O2 to decrease endogenous peroxidase, then incubated with primary antibody (antiBDNF 1:500, Alomone Labs) for 48 h at 4 °C, and biotinylated secondary antibody (1:200). Sections were stained using ABC Vectastain, HRP, and DAB. Sections were then placed onto slides, dehydrated and cleared, then coverslipped with Permount. The mean gray value (MGV) of the dentate gyrus, CA1, and CA3 was quantified using ImageJ freeware. To correct for differing background, the background gray scale value of an area adjacent to the area of interest was subtracted from the mean gray scale value.

2.3.2. Object location test of spatial memory Spatial memory was assessed using the Object Location Test (OLT), as developed by Ennaceur et al. (1997) and described by Ziegler and Thornton (2010). Each OLT consists of two trials, exposure and test, separated by an intertrial interval. In the exposure trial, each rat was introduced into the arena (with shavings) with two identical objects, each 20 cm from two adjacent corner walls. Behavior was considered exploratory when the nose of the rat was within 2 cm of the object. Time spent exploring each object was recorded over a 5 min period. Rats were then returned to their home cage for an intertrial interval of 30 min. Objects were cleaned and one object was moved to one of two configurations, counterbalanced to correct for any left-right preference. In the test trial, each rat was reintroduced to the arena equidistant from the two objects, and time exploring was recorded for 3 min. Although ovx rats generally show robust spatial memory with very short (e.g. 1 min) intertrial intervals, they fail to demonstrate strong spatial memory with an ITI of 30 min (Berry et al., 2008; Ziegler and Thornton, 2010). Each animal underwent 3 Object Location Tests, each separated by 4–6 days. A test was considered invalid if the rat failed to explore both objects or explored for < 10 s in either the exposure or test trial. Generally, approximately 2% of tests were considered invalid and all females had at least 2 valid tests. All valid tests were averaged for each rat.

2.6. Golgi impregnation Females were briefly anesthetized with isoflurane and brains were removed via rapid decapitation. Brains were rinsed in ice cold 10 mM PBS, blocked, and placed in solutions provided in the Rapid Golgi Stain Kit (FD NeuroTechnologies, Ellicott City, MD). Briefly, brains were stored in A/B mixture for 16–17 d (RT, in dark), then in Solution C for 48 h (0-4C, dark). They were then sliced on a vibratome (200 μ sections) in Solution C (0-4C) and mounted onto gelatin-coated slides. After drying for 1 d (RT, dark) sections were rinsed, stained with Solution D/ E, rinsed, and dehydrated through an ascending series of ethanols (50–100%). Slides were cleared in xylenes, coverslipped with Permount and allowed to dry for several weeks. Secondary basal dendrites and tertiary apical dendrites were analyzed blindly from pyramidal cells from the CA1 region of the dorsal hippocampus. Neurons in all areas were chosen for analyses as follows: (1) cell bodies and dendrites were well impregnated; (2) dendrites were clearly distinguishable from adjacent cells and continuous. Spines were counted under oil (100×) using a hand counter and dendritic length measured using the Olympus CellSens 1.16 software and an Olympus BX-41 microscope. Spine density was calculated by dividing spine number by the length of the dendrite and data expressed as number of spines/10 μm dendrite.

2.4. Western blot analysis Females were briefly anesthetized with isoflurane anesthetic and brains were removed via rapid decapitation. Brains were snap-frozen in isopentane and stored at −80C. A freezing microtome was used to expose the area corresponding to −1.80 posterior to bregma (Paxinos and Watson, 2006), and tissue from both sides of the medial dorsal hippocampus encompassing the DG, CA1, and CA3 regions was collected, using a 5 mm tissue punch to ensure constant volume. Medial dorsal hippocampus was chosen due to its established role in spatial memory in rats (Broadbent et al., 2004). Tissue was homogenized via sonication in 1% SDS and quantified for total protein concentration with the DC Protein Assay kit (Bio-Rad). 20 μg of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blocked for 2 h at room temperature in 5% BSA in phosphate-buffered saline with 0.1% tween20 (PBS-T). Then membranes were incubated in primary antibodies diluted in 5% BSA overnight at 4 °C: anti-BDNF (1:200, Santa Cruz N20) which detects both precursor and mature BDNF; anti-pTrkB (1:1000, generous gift from the Moses Chao lab); and anti-β-actin (1:5000, Sigma). Lastly, membranes were incubated in horseradish peroxidaselinked secondary antibodies diluted in PBS-T (1:5000), and imaged. Quantitative values of optical density were acquired with ImageJ

2.7. Statistical analysis Values are mean ± SEM and for all statistical tests an alpha level of ≤0.05 was considered statistically significant. For the test trial on the OLT, planned comparisons of the moved and unmoved object within a group were conducted using paired t-tests. Groups were considered to show good spatial memory if the amount of time spent exploring the moved object was statistically significantly greater than time spent on the unmoved object. Percent time spent investigating the moved object was compared across groups using a one-way ANOVA with individual planned comparisons using the Fisher's Least Significant Difference (LSD) test. Data for the exposure trials were analyzed using paired ttests comparing exploration times for left and right objects. Locomotor activity (lines crossed in 5 min period) was compared across groups using a one-way ANOVA and Tukey's post-hoc analysis if significance was reached. Western blot and immunohistochemical data were analyzed with one-way ANOVA and LSD tests. Golgi data were analyzed with ANOVA. Effect size was determined by Cohen's d for all pairwise comparisons and eta-squared (η2) for all ANOVA statistics. 3

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Fig. 2. Top: Preferential exploration of the moved object was not due to differential exploration during the exposure trial of the Object Location Test. There were no statistically significant differences in time spent exploring the left vs. right object in any of the groups. Bottom: There were no significant differences found between groups in the number of lines crossed during the locomotor activity test, either before or after drug treatment. N = 12–14 per group.

Fig. 1. Estradiol improved spatial memory, and the TrkB antagonist ANA-12 abrogated spatial memory in E-implanted ovx rats. Top: E + Veh rats showed good spatial memory, exploring the moved object significantly more than the unmoved (*p < 0.05). ANA-12 treatment led to deficits in spatial memory in E rats, with no significant difference between moved and unmoved exploration times (p > 0.05). Bottom: E animals spent a significantly larger percent of the time exploring the moved object than did Blank animals (*Blank + Veh vs. E + Veh, p < 0.05). E + ANA-12 females did not differ significantly from Blank + Veh females. N = 12–14 animals per group.

The percent time spent exploring the moved object further confirmed that the only treatment that significantly increased spatial memory was treatment with estradiol with intact BDNF signaling (Fig. 1). A one-way ANOVA revealed statistically significant differences in percentages between groups (F(2,37) = 4.25; p < 0.05; η2 = 0.077). The E + Veh group spent a statistically significantly higher percentage of time exploring the moved object compared to the Blank + Veh group (T(24) = 2.16; p < 0.05; d = 0.88). The E + ANA12 group did not differ significantly from the Blank + Veh group (T(24) = 0.30; p > 0.05; d = 0.06). These differences did not appear to be due to differential exploration of the objects during the exposure trial, or to differences in locomotor activity (Fig. 2). During the exposure trials there was so significant spatial bias for one of the two objects (p > 0.05 between left vs. right exploration times). There were no significant differences found between groups in the number of lines crossed during the locomotor tests. A one-way ANOVA revealed no statistically significant differences between groups in number of lines crossed either before treatment (F (2,37) = 2.61; p > 0.05; η2 = 0.15) or 4–6 h after treatment (F (2,37) = 0.11; p > 0.05; η2 = 0.01).

3. Results 3.1. Experiment 1: TrkB inhibition blocks estradiol's facilitation of spatial memory in ovx rats To investigate whether blocking BDNF-signaling would eliminate estradiol's facilitatory effects on spatial memory, female rats were ovx and implanted with either an estradiol (E) or blank capsule (Blank). Beginning four days later, rats were habituated to the test arena and then observed for locomotor activity. Females were then tested on the OLT three times with 4–6 d between tests. On each Object Location Test day, rats were injected with the TrkB antagonist ANA-12 (0.5 mg/kg) or vehicle, 4–6 h prior to spatial memory testing. Immediately after the last OLT another locomotion test was run and females were sacrificed. This experiment consisted of three groups: (1) blank implant and vehicle (Blank + Veh; n = 12); (2) estradiol implant and vehicle (E + Veh; n = 14); and (3) estradiol implant and TrkB antagonist ANA12 (E + ANA-12; n = 14). Blocking BDNF signaling with ANA-12 abrogated the beneficial effects of estradiol on spatial memory (Fig. 1). Estradiol-implanted ovx rats treated with vehicle showed robust discrimination between moved and unmoved objects (E + Veh: T(13) = 2.62; p < 0.001; d = 1.03), while blank-implanted animals treated with vehicle showed no significant preference between objects (Blank + Veh: T(11) = 1.83; p > 0.05; d = 0.78). Estradiol-implanted ovx rats treated with ANA-12 showed no significant preference between moved and unmoved object (E + ANA-12: T(13) = 1.56; p > 0.05; d = 0.61) indicating poor spatial memory.

3.2. Experiment 2: TrkB inhibition blocks Antide's facilitation of spatial memory in ovx rats To investigate whether a BDNF antagonist would block the enhancement of spatial memory by Antide, all rats were ovx, allowed to recover for 4d, then habituated to the arena, and tested for locomotor activity. Females were then tested on the OLT three times with 4–6 d between tests. On each OLT test day, rats were treated with either Antide (1 mg/kg) or vehicle and either the TrkB-antagonist ANA-12 (0.5 mg/kg) or vehicle, 4–6 h prior to testing. Immediately after the last 4

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Fig. 3. Antide's facilitation of spatial memory in ovx females was dependent on BDNF-TrkB signaling. Top: Ovx control (Veh + Veh) rats did not show strong spatial memory as they explored both objects equally. Antide + Veh rats showed good spatial memory, exploring the moved object significantly more than the unmoved (p < 0.05). ANA-12 treatment led to deficits in spatial memory in Antide rats, with no significant difference between moved and unmoved exploration times (p > 0.05). Bottom: There was a statistically significant difference in Antide animals compared to control in percent time spent exploring the moved object (*Veh + Veh vs. Antide + Veh, p < 0.05). N = 8–11 animals per group.

Fig. 4. Top: Preferential exploration of the moved object was not due to differential exploration during the exposure trial of the Object Location Test. There were no significant preferences for left vs. right object in any group. Bottom: There were no significant differences found between groups in the number of lines crossed during the locomotor activity test either before or after drug treatment. N = 8–11 animals per group.

groups in the number of lines crossed, a measure of locomotor activity. A one-way ANOVA revealed no statistically significant differences between groups in number of lines crossed either before testing (F (2,27) = 1.57; p > 0.05, η2 = 0.23) or after the final OLT (F (2,27) = 0.05; p > 0.05; η2 = 0.004).

OLT another locomotion test was run and females were sacrificed. This experiment consisted of three groups: (1) vehicle and saline (Veh + Veh; n = 8); (2) Antide and saline (Antide + Veh; n = 11); and (3) Antide and ANA-12 (Antide + ANA-12; n = 11). Blocking BDNF signaling with ANA-12 mitigated the beneficial effects of Antide treatment on spatial memory (Fig. 3). Rats treated with Antide and vehicle showed robust discrimination between moved and unmoved objects (Antide + Veh: T(10) = 2.43; p < 0.01; d = 1.08), while control animals treated with vehicle showed no significant preference between objects (Veh + Veh: T(7) = 2.62; p > 0.05; d = 0.01). Antide-treated rats injected with ANA-12 showed no significant preference between moved and unmoved object (Antide + ANA-12: T(10) = 1.34; p > 0.05; d = 0.60) indicating poor spatial memory. The percent time spent exploring the moved object further confirmed that the only treatment that significantly increased spatial memory was treatment with Antide (Fig. 3). A one-way ANOVA revealed statistically significant differences in percentages between groups (F(2,27) = 1.19; p < 0.001; η2 = 0.08). There was a statistically significant difference between Veh + Veh and Antide + Veh percentages (T(17) = 3.21; p < 0.05; d = 1.18). The Antide + ANA-12 group did not differ significantly from the Veh + Veh control females (T(17) = 1.66; p > 0.05; d = 0.20). These differences did not appear to be due to differential exploration of the objects during the exposure trial, or differences in locomotor activity (Fig. 4). During the exposure trial, there was no significant spatial bias for one of the two objects (p > 0.05 between left vs. right exploration times). There were no significant differences found between

3.3. Experiment 3: effects of E and Antide treatment on hippocampal immunoreactive BDNF levels To investigate the effects of estradiol or Antide on hippocampal expression of BDNF, a separate cohort of rats was ovx and implanted with either estradiol or a blank implant. On days 7 and 12 after ovx they were injected with either Antide (1 mg/kg) or vehicle and then sacrificed 4–6 h later. They did not undergo behavioral testing. This resulted in the following groups: (1) blank implant and vehicle (Blank; n = 4); (2) estradiol implant and vehicle (E; n = 5); (3) blank implant and Antide (Antide; n = 5). Five sections were averaged per brain. Both estradiol and Antide treatment increased BDNF expression throughout the DG, CA1 and CA3 regions of the hippocampus (Fig. 5). In the DG region, a one-way ANOVA found significant differences in staining levels between groups (F(2,11) = 13.51; p < 0.001; η2 = 0.74). LSD comparisons showed that the mean Blank gray value for staining in the DG differed significantly from E (T(7) = 5.69; p < 0.001; d = 4.33) and Antide (T(7) = 5.99; p < 0.001; d = 4.56) mean values. There were no significant differences between E and Antide (T(8) = 0.71; p > 0.05; d = 0.50). In the CA1 region, one-way ANOVA revealed similar differences between groups (F(2,11) = 6.33; p < 0.01; η2 = 0.58). LSD comparisons revealed significant differences between Blank values and all other groups (p < 0.05 across all comparisons). There were no significant differences between E and Antide (T(8) = 1.27; p > 0.05; d = 0.90). In the CA3 region, one-way ANOVA 5

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Fig. 5. Both estradiol and Antide increased immunoreactive BDNF expression in the hippocampus of ovx rats. Top: Representative BDNF stains in the hippocampus. Bottom: Treating ovx rats with estradiol (E) significantly increased BDNF expression, as measured by mean gray value, in the CA1, CA3, and the dentate gyrus (DG) of the hippocampus compared to control animals (Blank) (*p < 0.001 versus Blank within same brain region). Treatment with Antide increased BDNF expression in the hippocampus relative to Blank, in CA1, CA3, and DG (*p < 0.001 versus Blank within same brain region). N = 4–5 animals per group with 5 sections analyzed per animal.

revealed differences between groups (F(2,11) = 15.93; p < 0.001; η2 = 0.72). LSD comparisons revealed similar significant differences between Blank values and all other groups (p < 0.001 across all comparisons). There were no significant differences between E and Antide (p > 0.05; d = 0.98).

Fig. 6. Average Western blot absorbance values for brain-derived neurotrophic factor (BDNF), BDNF precursor protein (proBDNF), and phosphorylated tropomyosin receptor kinase B (pTrkB) in the dorsal hippocampus. Levels for all three proteins of interest were not significantly affected by estradiol (E) or Antide treatment. However, non-statistically significant increases were seen (p = 0.11) in BDNF and proBDNF protein levels in Antide-treated ovx females versus Blank. All bands were compared to β-actin loading control and quantified relative to the Blank band on each blot. N = 8 per group. Bottom: Representative blots for all proteins probed.

3.4. Experiment 4: assessing effects of E and Antide treatment on hippocampal BDNF, proBDNF, and pTrkB levels via Western blotting In order to elucidate the role of LH in BDNF-TrkB signaling, hippocampal tissue was examined for expression levels of BDNF, the precursor protein proBDNF, and its activated receptor, phosphorylated TrkB (pTrkB). Immediately after the last OLT of Experiments 1 and 2, rats from the Blank + Veh (n = 8), E + Veh (n = 8), and Antide + Veh (n = 8) groups were sacrificed, brains collected, and prepared for Western blotting. Across all proteins, no significant differences in expression levels were detected between groups. (Fig. 6). A one-way ANOVA for all proteins showed no significant differences in expression across groups (BDNF: F(2,21) = 0.98; p > 0.05; η2 = 0.10. proBDNF: F (2,21) = 0.91; p > 0.05; η2 = 0.09. pTrkB: F(2,21) = 0.38; p > 0.05; η2 = 0.04). LSD tests found a trend toward a non-statistically significant increases in BDNF expression in Antide versus Blank (T(14) = 1.65, p = 0.11, d = 0.88) and proBDNF expression in Antide versus Blank (T(14) = 1.59; p = 0.11; d = 0.85).

tested for locomotion. They were then tested on the OLT, three times, with 4–6 d between tests. On each test day either Antide (1 mg/kg) or vehicle was injected 4–6 h prior to behavioral testing. Immediately after the last behavioral test, they underwent another locomotion test and brains were processed for Golgi impregnation. This resulted in three groups: (1) Blank + Veh (n = 6); (2) E + Veh (n = 8); and Blank + Antide (n = 10). Consistent with previous studies ovx females did not show significant spatial memory whereas both E and Antide treated females showed robust spatial memory. That is, Blank + Veh did not spend more time on the moved vs unmoved object (9.67 ± 1.73 s unmoved, 8.58 ± 0.85 s moved, T(5) = 1.28; p > 0.05; d = 0.33). Both E + Veh and Blank + Antide females spent significantly more time exploring the moved object (E unmoved = 7.57 ± 1.41 s and moved = 19.2 ± 1.82 s, T(7) = 6.38; p < 0.001; d = 2.53; Antide unmoved = 6.35 ± 0.61 s vs moved = 18.6 ± 2.1 s, T(9) = 5.85; p < 0.0001; d = 2.52). Six females from each treatment group were randomly chosen for Golgi analysis. Six cells per region/brain were quantified. A one-way ANOVA revealed no significant differences between any of the

3.5. Experiment 5: no effect of E or Antide on hippocampal CA1 spine density To ascertain whether Antide affects spine density in the hippocampus, another cohort of rats was ovx and implanted with either a blank or E capsule, allowed to recover for 4 d, then habituated and 6

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BDNF mRNA in whole hippocampus whereas Singh et al. (1995) administered E after 28 wk of ovx and saw increases in BDNF mRNA in some regions of hippocampus but not in CA1. As for effects of LH on BDNF, Palm et al. (2014) found that administration of leuprolide acetate (a GnRH super agonist that lowers LH levels) increases BDNF mRNA in the prefrontal cortex, however data were not reported for the hippocampus. Collectively, these findings are consistent with the notion that estradiol and Antide may increase hippocampal BDNF mRNA levels and perhaps the processing of pro-BDNF into the mature form of BDNF. This may be beneficial as pro-BDNF and BDNF can have opposing effects on neuronal physiology and plasticity and pro-BDNF may compromise memory (Buhusi et al., 2017). Additionally, levels of activated receptor phosphoTrkB protein did not significantly change after either estradiol or Antide treatment. In contrast, Pan et al. (2010) saw increased TrkB mRNA levels in whole hippocampi in rats fed dietary estradiol for 12 wk and Spencer et al. (2008) saw increased pTrkB immunoreactivity in proestrous mice using IHC. However, neither of these studies measured phosphorylated/active protein so it is unclear if the increased TrkB mRNA resulted in active, phosphorylated TrkB receptor. Alternatively, differences may be due to variations in hormone administration or some other factor. Furthermore, no change in dendritic spine density on pyramidal cells in CA1 was seen in any group. Estradiol injection has been shown to increase dendritic spine density both chronically and acutely in ovx rats in CA1 (Frankfurt and Luine, 2015). It is unclear why the present results differ from earlier studies, but one possibility is that estradiol exposure was longer than in earlier studies where 5 μg of E was given for 48 h. Alternatively, these results suggest that spine density does not always need to be increased at the time of behavioral testing in order to see enhanced spatial memory. This is consistent with a previous study that found that estradiol benzoate increased spine density in behaviorally naïve female rats but not in females tested on a water maze (Frick et al., 2004). In contrast, in a study in adolescents, 11 days of estradiol injections increased spine density in CA1in both behaviorally tested and non-tested females (Bowman et al., 2019). Taken together it appears as though timing of estradiol administration and behavior influence fluctuations in spine density. These results expand our knowledge on the cellular mechanism of action of LH in the CNS. LH was shown to bind LH receptors in immortalized hippocampal neurons to stimulate the production of the important second messenger cyclic adenosine monophosphate (cAMP; Zhang et al., 1999). LH treatment in neuronal cultures also led to increased expression of extracellular-signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK), both of which regulate cAMP levels (Meng et al., 2007). cAMP acts on several pathways associated with memory consolidation and plasticity, however the manner in which LH might impair consolidation through these mechanisms remains unclear. Leuprolide acetate, another LH-lowering treatment, was found to increase mRNA levels of several molecules involved in synaptic plasticity in the prefrontal cortex, including BDNF, beta-catenin, and pCamKII, all molecules that are associated with synaptic plasticity and memory (Palm et al., 2014). Surprisingly, direct application of LHhomologue hCG on hippocampal cultures from d18 embryos led to increases in higher order neurites and branch points, potentially indicating higher synaptic plasticity (Blair et al., 2019). These authors did not report any spine data in dorsal hippocampus but saw alterations in branching in retrosplenial cortex of adult mice, indicating potential changes in neural networks that may be independent of spine density changes. Nevertheless, more research is needed for a better understanding of LH signaling in the CNS. Future studies should also elucidate the exact signaling cascades that LH stimulates in the hippocampal neurons, as this will present a more complete picture of the various pathways that LH may activate or inactivate in the brain to influence spatial memory. The current experiments provide evidence that estradiol and the LHlowering drug, Antide, improve spatial memory by increasing

Table 1 Dendritic spine density for basal and apical dendrites in CA1. Data expressed as the mean ± SEM per 10 μm dendrite. N = 6 per group. One-way ANOVA indicated no significant differences between groups.

Basal Apical

Blank + Veh

Estradiol + Veh

Blank + Antide

11.31 ± 0.03 13.5 ± 0.04

11.22 ± 0.05 12.37 ± 0.06

11.56 ± 0.08 12.61 ± 0.04

treatments on spine density in CA1 (see Table 1; basal dendrites F (2,15) = 0.48; p > 0.05; η2 = 0.01; apical dendrites F(2,15) = 1.24; p > 0.05; η2 = 0.16). 4. Discussion These results establish an important relationship between estradiol, LH, and BDNF-TrkB signaling. Blocking the BDNF-TrkB signaling pathway with TrkB antagonist ANA-12 inhibited spatial memory in rats treated with estradiol or LH-lowering treatment (Antide). Immunohistochemistry data showed that estradiol and Antide increased BDNF expression in the hippocampus. These findings suggest that the spatial memory enhancement caused by lowering LH levels depends upon an intact BDNF-TrkB signaling pathway. The ablation of spatial memory by ANA-12 in rats treated with estradiol or Antide was most likely due to ANA-12's action at the TrkB receptor. In the current study, females were tested 4–6 h after ANA-12 treatment. Cazorla et al. (2011) characterized ANA-12's biochemical specificity for TrkB receptors and found that the drug was still bioavailable after 6 h. Therefore ANA-12 likely blocked BDNF-signaling in ovx rats treated with estradiol or Antide, leading to inhibited spatial memory. Other studies have used ANA-12 to block hippocampus dependent behaviors: increases in spatial memory due to treatment with Telmisartan, an angiotensin II receptor blocker, were abolished when TrkB activity was blocked via ANA-12 (Kishi et al., 2012), and the antidepressant-like effect of 7,8-DHF was ablated by ANA-12 (Zhang et al., 2014). However, the present findings are the first to show that the behavioral effects of hormones on memory, specifically estradiol and LH, were modulated by blocking BDNF-TrkB signaling. In all behavioral trials, the demonstration of spatial memory (i.e. a preference for the moved versus the unmoved object) was unlikely to be due to differences in exploration levels, side preference, or locomotion levels. In the exploration trial, there were no significant differences in exploration of left and right objects. All OLT test trials were also counterbalanced to correct for any possible side bias. The results of the open-field locomotion test also suggested no differences in locomotion levels between groups. There was a non-statistically significant trend toward greater exploration of the moved object by the control (Blank + Veh) females in Expt1 (p = 0.07) but this was not replicated in subsequent experiments (i.e. Veh + Veh in Expt 2, p = 0.25 or Blank + Veh in Expt 5, p = 0.58) so it was likely random variation. Using immunohistochemistry (IHC), we observed significant increases in BDNF expression in the CA1, CA3, and dentate gyrus of the hippocampus after either estradiol or Antide treatment. Using Western blotting, by contrast, we observed a trend toward an increase in BDNF but saw no statistically significant changes in total protein levels of BDNF or proBDNF in the medial dorsal hippocampus after either estradiol or Antide treatment. We had anticipated that increases in BDNF would be observed with both IHC and Western blotting. It may be that the medial hippocampal punches were too small to reliably measure differences. However, use of whole hippocampi has also resulted in contradictory findings. Pan et al. (2010) fed female rats with dietary estrogen for 12 wk and found increased total BDNF protein in whole hippocampus. Contrastingly, Gibbs (1999) exposed ovx females to estradiol for 48 h and observed decreased BDNF protein in whole hippocampus. Both Gibbs (1999) and Pan et al. (2010) saw increased 7

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expression levels of BDNF in the hippocampus. Our findings suggest that Antide may improve cognition in ovx rats by lowering levels of LH which in turn increases levels of BDNF, which then interacts with TrkB to activate pathways involved in spatial memory consolidation. However, it is important to note that these data do not prove that estradiol and/or Antide interact directly with BDNF/TrkB to influence spatial memory. It is possible that they act via a more indirect mechanism. Moreover, the behavioral results indicate that although BDNF signaling is essential for spatial memory formation, the spine density results indicate that in this model the enhancement is probably not mediated by dendritic spine formation in hippocampal CA1. Understanding the complete mechanism of effects exerted by estradiol and Antide, and by extension the effects of LH in the CNS, will provide a clearer picture of the hormonal changes that factor into age-related memory changes and cognitive decline. Moreover, these findings may pave the way for the development of more effective therapeutics for AD and other debilitating cognitive disorders.

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Funding sources This work was supported by a grant-in-aid from Oberlin College, with additional grant support from the Nu Rho Psi Honor Society and the Robert Rich Student Research Fund of Oberlin College. Acknowledgements The authors thank Kate Van Pelt and Alex Jabbour for their assistance in Western blot assays. Additionally the authors thank the Moses Chao lab of New York University for their generous donation of the anti-phosphoTrkB antibody used in these studies. Declaration of competing interest None. References Berry, A., Tomidokoro, Y., Ghiso, J., Thornton, J., 2008. Human chorionic gonadotropin (a luteinizing hormone homologue) decreases spatial memory and increases brain amyloid-beta levels in female rats. Horm. Behav. 54, 143–152. https://doi.org/10. 1016/j.yhbeh.2008.02.006. Bimonte, H.A., Denenberg, V.H., 1999. Estradiol facilitates performance as working memory load increases. Psychoneuroendocrinology 24, 161–173. https://doi.org/10. 1016/S0306-4530(98)00068-7. Bimonte-Nelson, H.A., Nelson, M.E., Granholm, A.C., 2004. Progesterone counteracts estrogen-induced increases in neurotrophins in the aged female rat brain. NeuroReport 15, 2659–2663. https://doi.org/10.1097/00001756-20041203000021. Blair, J.A., Palm, R., Chang, J., McGee, H., Zhu, X., Wang, X., Casadesus, G., 2016. Luteinizing hormone downregulation but not estrogen replacement improves ovariectomy-associated cognition and spine density loss independently of treatment onset timing. Horm. Behav. https://doi.org/10.1016/j.yhbeh.2015.10.013. Blair, J.A., Bhatta, S., Casadesus, G., 2019. CNS luteinizing hormone receptor activation rescues ovariectomy-related loss of spatial memory and neuronal plasticity. Neurobiol. Aging 78, 111–120. https://doi.org/10.1016/j.neurobiolaging.2019.02. 002. Bowman, R.E., Hagedorn, J., Madden, E., Frankfurt, M., 2019. Effects of adolescent Bisphenol-A exposure on memory and spine density in ovariectomized female rats: adolescence vs adulthood. Horm. Behav. 107, 26–34. https://doi.org/10.1016/j. yhbeh.2018.11.004. Broadbent, N.J., Squire, L., Clark, R.E., 2004. Spatial memory, recognition memory, and the hippocampus. PNAS 101, 14515–14520. https://doi.org/10.1073/pnas. 0406344101. Buhusi, M., Etheredge, C., Granholm, A.C., Buhusi, C.V., 2017. Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice. Front Aging Neurosci. 9, 284. https://doi.org/10.3389/fnagi.2017.00284. Burnham, V., Thornton, J., 2015. Luteinizing hormone as a key player in the cognitive decline of Alzheimer's disease. Horm. Behav. 76, 48–56. https://doi.org/10.1016/j. yhbeh.2015.05.010. Burnham, V., Sundby, C., Laman-Maharg, A., Thornton, J., 2017. Luteinizing hormone acts at the hippocampus to dampen spatial memory. Horm. Behav. 89, 55–63. https://doi.org/10.1016/j.yhbeh.2016.11.007. Casadesus, G., Webber, K.M., Atwood, C.S., Pappolla, M.A., Perry, G., Bowen, R.L., Smith, M.A., 2006. Luteinizing hormone modulates cognition and amyloid-beta deposition

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