Behavioural Brain Research 262 (2014) 68–73
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Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats Wayne R. Hawley a,∗ , Elin M. Grissom a , Nicole M. Moody b , Gary P. Dohanich a,b , Nandini Vasudevan b,c a
Department of Psychology, Tulane University, New Orleans, LA 70118, United States Program in Neuroscience, Tulane University, New Orleans, LA 70118, United States c Department of Cellular and Molecular Biology, Tulane University, New Orleans, LA 70118, United States b
h i g h l i g h t s • Estradiol treatment enhanced spatial recognition memory in ovariectomized rats. • G-1 treatment enhanced spatial recognition memory in ovariectomized rats. • Neither estradiol nor G-1 impacted levels of activity in ovariectomized rats.
a r t i c l e
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Article history: Received 17 November 2013 Received in revised form 4 January 2014 Accepted 7 January 2014 Available online 18 January 2014 Keywords: Estradiol Ovariectomy G-protein-coupled receptor 30 G-1 Spatial learning and memory
a b s t r a c t In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the ␣ or  isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 g) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 g) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Elevated levels of ovarian hormones impact performance on a variety of spatial learning and memory tasks [1–6], including those that take advantage of the tendency of rodents to seek out spatial novelty. These tasks include object location tasks [7,8] and a Ymaze task [9], two hippocampus-based cognitive tasks that are free
∗ Corresponding author at: Tulane University, Department of Psychology, 2007 Percival Stern Hall, New Orleans, LA 70118, United States. Tel.: +1 504 865 5331; fax: +1 504 862 8744. E-mail addresses: [email protected], [email protected] (W.R. Hawley). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2014.01.006
from the stress associated with water escape and the motivation to obtain food reward [10–12]. Paralleling the memory enhancement observed during proestrus, when levels of ovarian hormones are naturally elevated, ovariectomized rodents treated with estradiol also exhibited an enhancement in spatial learning and memory on a variety of tasks [13–18]. However, it is important to keep in mind that the effects of estradiol treatment vary as a function of strain, dose and behavioral testing paradigm [1]. For instance, treatment of ovariectomized rodents with lower doses of estradiol, which mimic the peak in physiological levels that characterize proestrus, tend to facilitate spatial learning and memory, whereas higher doses have been shown to impair performance [16,17,19–22]. As would be expected, the enhanced spatial learning and memory in ovariectomized rodents following estradiol treatment
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involves the classic estrogen receptor ␣ and  isoforms. For example, genetic deletion of estrogen receptor  abrogated the spatial memory enhancement that occurred in ovariectomized rodents treated with estradiol [23]. Alternatively, lentivirus mediated overexpression of estrogen receptor ␣ in the hippocampus [24,25], or administration of selective estrogen receptor ␣ or  modulators was sufficient to enhance spatial cognition in ovariectomized rats and mice [18,26–29]. In addition to the classic estrogen receptor isoforms, the memory enhancing effects of estradiol on spatial learning and memory are modulated by activation of the putative estrogen membrane receptors GPR30, which are located in the hippocampus, as well as other brain structures [30,31]. In ovariectomized rats, treatment with G-1, a specific agonist for estrogen membrane receptor GPR30, mimicked the enhancement in spatial learning that occurred on a delayed matching-to-position T-maze task following treatment with either estradiol, or selective agonists for estrogen receptor ␣ or  [27]. Alternatively, treatment with G-15, which is a GPR30 antagonist, abrogated the enhancing effects of estradiol treatment to ovariectomized rats on the same delayed matching-to-position T-maze task [32]. Therefore, because treatment of ovariectomized rats with G-1 mimicked the effects of estradiol treatment on a food-motivated spatial learning task [27], we predicted that G-1 treatment would enhance spatial recognition memory on a Y-maze task, an effect we expected to be paralleled following treatment with a low dose of estradiol.
2. Materials and methods 2.1. Animals All procedures, which were identical for both experiments, were approved by the Tulane University Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Female Long-Evans rats in Experiment 1 (N = 44) and Experiment 2 (N = 20) were purchased from Harlan Laboratories Inc. (Indianapolis, IN) and arrived at the animal care facility at Tulane University at approximately 60 days of age. Rats were housed in pairs, provided ad libitum access to food and water (Teklad Inc., Madison, WI), and maintained on a reversed 12:12 h light-dark cycle (lights off at 11:00 h) in animal care facilities at Tulane University accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Rats were acclimated to the vivarium for 5–7 days upon arrival to the facility before procedures began. In addition, rats were acclimated to procedural and behavioral testing rooms for at least 30 min prior to procedures or behavioral testing, which occurred during the dark phase of the light–dark cycle.
2.2. Ovariectomy At approximately 65 days of age, rats were ovariectomized under anesthesia induced by intraperitoneal injections of ketamine (100 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (7 mg/kg, Miles Laboratories, Shawnee, KS). Dorsal ventral incisions were made bilaterally approximately 5 cm from the most posterior point of the rib cage. After the ovaries were excised, ovarian blood vessels were ligated, the muscle wall was closed with 4.0 silk sutures (Teleflex Medical, Kenosha, WI), and the skin was closed with titanium wound clips (Mercer Glassware Inc., New York, NY), which were removed 10 days after surgery. Rats were provided free access to drinking water containing ibuprofen (25 mg/kg) to reduce post-surgical pain during their recovery for 3 consecutive days after surgery. Then, to acclimate to experimenters, rats were
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handled for 1 min each day for 7 consecutive days beginning 1 week after surgery. 2.3. Hormone (Experiment 1) and drug treatment (Experiment 2) On the last 2 days of handling, which occurred 48 and 24 h prior to beginning behavioral testing, rats in Experiment 1 were administered intramuscular injections of either sesame oil (0.1 ml; n = 10; Sigma–Aldrich Co., St. Louis, MO) or 17-estradiol benzoate (1 g, n = 12; 10 g, n = 10; or 25 g, n = 12; Sigma–Aldrich Co.) suspended in sesame oil (0.1 ml). Rats in Experiment 2 were administered intramuscular injections of either sesame oil (0.1 ml; Sigma–Aldrich Co.) or G-1 (25 g; Sigma–Aldrich Co.), the estrogen membrane receptor agonist, suspended in sesame oil (0.1 ml). The dose of G-1 was selected based on preliminary studies and findings which indicated that higher doses of specific estrogen receptor agonists are often necessary to mimic the effects of estradiol on spatial learning [18,28] and on the morphology of the hippocampus [17,29]. 2.4. Y-maze task Spatial recognition memory was characterized on a hippocampus-based version of a Y-maze task [10]. The maze was constructed from gray opaque Plexiglas that formed three identical arms (50 × 10 × 20 cm; Stoelting ANY-maze, Wood Dale, IL), and was surrounded by a variety of two and three dimensional extra-maze cues. To minimize the amount of time experimenters spent in proximity to the Y-maze, the start arm was placed in the testing environment in the position that provided the most efficient exit for experimenters. For both experiments, all rats were entered into the maze from the same start arm position within the testing environment. Rats were placed into the maze for a 15-min information trial and were allowed to freely explore the start arm and a second arm, but access to the third arm, which was counterbalanced between conditions, was blocked by an opaque plastic partition. After a delay period of 48 h, rats were placed back into the same start arm and allowed to freely explore all three arms for a 5-min retention trial, which was video recorded by and overhead camera for later scoring. The maze was cleaned thoroughly with 10% ethanol and air-dried after each trial to remove olfactory cues. Entry into an arm was defined as all four paws crossing into the arm proper by an experimenter blind to the conditions. On the retention trial, spatial recognition memory was indicated by calculating a novel arm preference index, which was the average of the percentage of entries and time into the arm associated with the novel environment relative to all of the arms. Greater preference for the arm associated with the novel environment indicated better spatial recognition memory. The total number of arms entered during the retention trial served as an indicator of general activity. However, because rats can quickly acclimate to novelty while exploring a Y-maze [33], spatial memory and activity were assessed for both the first minute, as well as the entire five minutes of the retention trial. 2.5. Statistical analyses Analyses of variance and independent samples t-tests were conducted to examine differences in activity between conditions on the retention trial in Experiment 1 and Experiment 2, respectively. One-sample t-tests (relative to chance, 33%) were conducted to determine within-group preference for the arm associated with the novel environment on the retention trial of the Y-maze task, which is an indicator of spatial recognition memory [34]. Statistical significance was indicated by p ≤ 0.05. Rats that failed to move from
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A Novel Arm Composite Score (%T+%E/2)
66 55 44
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Vehicle EB (1 µg) EB (10 µg) EB (25 µg)
33 22 11
A
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Novel Arm Composite Score (%T+%E/2)
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***
44 33 22 11
Vehicle EB (1 µg) EB (10 µg) EB (25 µg)
1st min
1st min
All 5 min
B Total Arm Entries
Total Arm Entries
18 16 14 12 10 8 6 4 2 0
Vehicle G-1 (25 µg)
0
0
B
p = 0.06
Fig. 1. Effects of estradiol on activity and spatial recognition memory on the retention trial of a Y-maze task. (A) Only rats administered 1 g of estradiol 24 and 48 h prior to the information trial exhibited recognition of the novel arm during the first minute of the retention trial, as indicated by a preference for the novel arm relative to chance. (B) No differences in activity, as indicated by total arm entries, were found as a function of estradiol treatment. *p ≤ 0.05 relative to chance performance, 33%.
the start arm, as well as those that climbed on top of the walls of the Y-maze during either the information or retention trial were excluded from analyses. 3. Results 3.1. Experiment1: the effects of estradiol on spatial recognition memory As depicted in Fig. 1A, rats administered 1 g [t(6) = ± 2.39, p ≤ 0.05] of estradiol 48 and 24 h prior to the information trial of the Y-maze task, but not those administered oil [t(7) = ± 4.30, p ≤ 0.01], exhibited a preference for the arm associated with the novel environment during the first minute of the retention trial. Neither administration of 10 g [t(7) = ± 1.84, p = 0.11] nor 25 g [t(9) = ± 0.00, p = 1.00.] of estradiol increased preference for the arm associated with the novel environment during the first minute of the retention trial (Fig. 1A). Neither oil nor any of the doses of estradiol impacted preference for the arm associated with spatial novelty across the entire five minutes of the retention trial (Fig. 1A). Importantly, no differences in activity, as indicated by total arm entries on the retention trial, were found between rats administered oil and those administered estradiol (Fig. 1B). Therefore, as indicated during the early stage of the retention trial, a low dose of estradiol enhanced spatial recognition memory without impacting activity. 3.2. Experiment 2: the effects of the GPR30 agonist G1 on spatial recognition memory The results illustrated in Fig. 2A indicated that rats treated with G-1 prior to the information trial tended to exhibit a preference
Vehicle G-1 (25 µg)
18 16 14 12 10 8 6 4 2 0 1st min
All 5 min
All 5 min
All 5 min
Fig. 2. Effects of the estrogen membrane receptor agonist G-1 on activity and spatial recognition memory on the retention trial of a Y-maze task. (A) Only rats administered G-1 24 and 48 h prior to the information trial exhibited recognition of the novel arm, as indicated by a greater preference for novel arm relative to the familiar arm across the entire 5 min of the retention trial. (B) No differences in activity, as indicated by total arm entries, were found between rats administered oil or G-1. *p ≤ 0.05 relative to chance performance, 33%.
for the arm associated with the novel environment during the first minute of the retention trial [t(6) = ± 2.37, p = 0.06]. However, as the retention trial progressed, rats treated with G-1 exhibited a preference for the arm associated with the novel environment as indicated across the entire five minutes of the retention trial [t(6) = ± 6.61, p < 0.01]. Rats treated with oil did not exhibit a preference for the arm associated with the spatial novelty during the first minute [t(8) = ± 1.60, p = 0.15] or the entire five minutes [t(8) = ± 0.39, p = 0.71] of the retention trial (Fig. 2A). As depicted in Fig. 2B, no differences in activity, as indicated by total arm entries on the retention trial, were found between rats administered the estrogen membrane receptor agonist G-1 and those administered oil 24 and 48 h prior to the information trial. Therefore, in ovariectomized rats, administration of a GPR30 agonist was sufficient to enhance spatial recognition memory without impacting activity. 4. Discussion The GPR30 agonist, G1, enhanced spatial memory on the retention trial of a Y-maze task when administered to ovariectomized rats at 48 and 24 h before the information trial. Treatment with a low dose of estradiol at 48 and 24 h prior to the information trial of a Y-maze task similarly enhanced spatial recognition memory in ovariectomized rats on the retention trial conducted 48 h later. Because there were no differences in activity level, which was indicated by the total number of arm entries on the retention trial, it is reasonable to propose that the preference for the arm associated with spatial novelty that emerged as a function of estradiol treatment in Experiment 1, and administration of G-1 in Experiment 2, was not impacted by the non-mnemonic factor of activity.
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Collectively, the results of Experiments 1 and 2 indicated that the memory enhancing effects of estradiol in ovariectomized rats on a spatial task that takes advantage of the preference of rats to seek out novelty also occurs following treatment with G-1, a drug that selectively activates the putative estrogen membrane receptor GPR30.
4.1. Ovarian hormones and spatial cognition During proestrus, when ovarian hormones are naturally elevated, gonadally intact rats exhibit enhanced spatial cognition on a variety of tasks motivated by the preference of rats to gravitate toward novelty [7–9,35], an effect which also occurs in ovariectomized rodents following administration of estradiol [18]. Moreover, similar to both the low dose (1 g) and timing of estradiol treatment that enhanced memory for spatial novelty in Experiment 1, administration of estradiol (4.5 g/kg) at 48 and 24 h prior to training also has been shown to enhance learning on a foodmotivated version of a hippocampus-based place learning task [19]. Therefore, in ovariectomized rats, low dose regimens of estradiol treatment, which are designed to approximate the physiological levels that occur during proestrus, result in enhanced cognition on a variety of spatial learning and memory tasks. However, with this in mind, it is important to note that in the current study, levels of estradiol were not measured out of concern that the stress associated with blood sampling could have impacted the acquisition or consolidation of spatial information at the time of the information trial. In addition, examining levels of estradiol following completion of the retention trial, which would have been 72-h after the last injection, would have provided little information as to what levels were present during the information trial, when spatial information was being learned. Unlike tasks that rely upon detection of spatial novelty, the effects of ovarian hormones on both water maze tasks [2,4] and food-motivated radial-arm maze tasks [6,36] have been somewhat inconsistent, such that in some instances rats in proestrus exhibited poorer spatial learning and memory [3,37]. Arguably, the inconsistent results of ovarian hormones on water maze tasks may have been due to elevated levels of stress hormones [3,5], which have been shown to interfere with spatial cognition [38]. This interpretation is important to consider given the hyper-responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis during proestrus, an effect that has been attributed to estradiol [39]. Therefore, as may be the case for examining the effects of sex steroids on spatial cognition in male rats [34], behavioral tasks that are contingent upon the preference of rats to seek out novelty, and not by the stress associated with water escape, may be more suitable for examining the relationship between estradiol and spatial cognition in female rats. Neither estradiol nor G-1 treatment impacted the nonmnemonic factor of exploratory behavior, which was indicated by the total number of arm entries during the retention trial. Furthermore, across both experiments, only one rat, which was not included in the analyses, failed to move from the start arm during the information trial, which indicated that neither estradiol nor G-1 impacted the tendency to explore novelty. In accordance with these findings, systemic administration of G-1 has been shown not to impact anxiety-like behavior in ovariectomized rodents [40]. Despite these findings, as well as results that indicate greater preference for the novel arm of a Y-maze is an indicator of hippocampus-based spatial memory [10], we cannot entirely rule out the possibility that treatment with either estradiol or G-1 merely impacted the tendency to explore novelty during the retention trial, and not just memory for novelty.
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4.2. Estrogen receptors and spatial cognition In Experiment 2, activation of the putative estrogen membrane receptor GPR30 in ovariectomized rats following treatment with G-1 resulted in an enhancement in spatial recognition memory. Following administration of estradiol conjugated to cyclodextrin, which activates estrogen-associated membrane receptors, ovariectomized rodents exhibited enhanced performance on a spatial version of the water maze task [41] and on object memory tasks, which characterize spatial and non-spatial memory [42,43]. However, only two other studies, both from the same laboratory, have directly examined the relationship between GPR30 and spatial cognition in rats [27,32]. Treatment of ovariectomized rats with G-1 for over 1 month resulted in enhanced spatial learning on a foodmotivated version of a delayed matching-to-position T-maze task, an effect that mimicked the beneficial effects of estradiol treatment [27]. In a follow-up study, treatment of ovariectomized rats for over 1 month with a GPR30 antagonist abrogated the enhancing effects of simultaneous estradiol treatment on the same spatial task [32]. Therefore, the results from Experiment 2 indicated that short-term treatment with G-1, which mimicked the short-term treatments with estradiol used in Experiment 1 and elsewhere [19,22], was sufficient to enhance spatial cognition in ovariectomized rats on a task not motivated by food reward. Although the results of Experiment 2 illustrate a role for GPR30 in spatial recognition memory, the contributions of estrogen receptors ␣ and  to spatial recognition memory remain to be determined. However, administration of agonists that selectively activate either estrogen receptor ␣ or  have been show to enhance performance on object placement tasks, which also rely on detection of spatial novelty [8,18,23]. Furthermore, when levels of estradiol are elevated, expression of estrogen receptor  was necessary for enhancements in spatial cognition to occur on an object location task [23] and a spontaneous spatial alternation task [35]. Therefore, in addition to GPR30, both the estrogen receptor ␣ and  isoforms impact learning and memory on tasks that rely upon the detection of spatial novelty. 4.3. Possible mechanisms mediating effects of estradiol and G-1 on spatial memory The memory enhancing effects of estradiol in Experiment 1 emerged during the first minute of the retention trial, whereas the beneficial effects of G-1 in Experiment 2 were observed across the entire 5-min of the retention trial. As suggested by Wright and colleagues [33], rats can quickly acclimate to a Y-maze, which may explain why rats treated with estradiol in Experiment 1 exhibited a preference for the novel arm only during the first minute. However, it also is important to consider how the distribution of specific estrogen receptor subtypes within the hippocampus, a brain structure that modulates spatial recognition memory [10], may have impacted the time course in novel arm preference as a function of estradiol and G-1 treatment. GPR30 is seemingly expressed to a greater extent in the rat hippocampus than estrogen receptor ␣ and  [30,44,45]. Interestingly, estrogen receptor  has been shown to repress transcriptional processes induced by estrogen receptor ␣ [46]. Although the interactive nature of GPR30 and estrogen receptors ␣ and  remains to be determined, interactions between estrogen receptor ␣ and  isoforms can subsequently impact the influence of estradiol on spatial cognition [47]. Conceivably, in the current study, treatment with estradiol, which activates all estrogen receptor subtypes, may have altered the beneficial effects of GPR30 activation. The reported effects of gonadal steroids, including estradiol, on spatial cognition are typically modest in unmanipulated rodents [1,48]. Prominent effects of estradiol emerge when performance
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has been compromised, for example, when cholinergic function is impaired by administration of agents designed to mimic the pathology associated with Alzheimer’s disease [49,50]. In addition to the hippocampus, GPR30 also is expressed in the basal forebrain, a brain region where administration of G-1 has been shown to mimic the stimulatory effects of estradiol on potassium-stimulated acetylcholine release within the hippocampus [31]. Interestingly, short-term treatment of ovariectomized rats with estradiol, which mimicked the time-course of estradiol treatment in Experiment 1 and G-1 administration in Experiment 2, elevated levels of acetylcholine release in the hippocampus during learning on a spatial task [51]. Furthermore, in ovariectomized rats, estradiol-induced up-regulation of synaptic spines in the hippocampus was contingent upon cholinergic innervation from the basal forebrain [52]. Given the increase in dendritic spine densities that occurs in the hippocampus of ovariectomized rodents following a single treatment with even a very low dose of estradiol [17], it is reasonable to believe that in addition to greater acetylcholine release [51] there also was an increase in synaptic connectivity during learning in rats treated with either estradiol or G-1. Arguably, the spatial recognition memory enhancement that occurred in rats treated with either estradiol or G-1 was mediated by activation of GPR30 in the basal forebrain, such that activation of GPR30 in the basal forebrain elevated levels of acetylcholine release in the hippocampus [31], which resulted in increased synaptic connectivity and enhanced neural communication during learning. Role of funding sources This research was supported by a National Foundation of Science CAREER award (#1053716) and Flowerree Summer Fellowships. Acknowledgement The authors gratefully acknowledge the expert supervision of animal care provided by Kimberly Scamardo. References [1] Dohanich G. Gonadal steroids, learning, and memory. In: Pfaff D, Arnold A, Etgen A, Fahrback S, Rubin R, editors. Hormones, brain and behavior. San Diego: Academic Press; 2002. p. 265–327. [2] Berry B, McMahan R, Gallagher M. Spatial learning and memory at defined points of the estrous cycle: effects on performance of a hippocampaldependent task. Behav Neurosci 1997;111:267–74. [3] Warren SG, Juraska JM. Spatial and nonspatial learning across the rat estrous cycle. Behav Neurosci 1997;111:259–66. [4] Frick KM, Berger-Sweeney J. Spatial reference memory and neocortical neurochemistry vary with the estrous cycle in C57BL/6 mice. Behav Neurosci 2001;115:229–37. [5] Rubinow MJ, Arseneau LM, Beverly JL, Juraska JM. Effect of the estrous cycle on water maze acquisition depends on the temperature of the water. Behav Neurosci 2004;118:863–8. [6] Pompili A, Tomaz C, Arnone B, Tavares MC, Gasbarri A. Working and reference memory across the estrous cycle of rat: a long-term study in gonadally intact females. Behav Brain Res 2010;213:10–8. [7] Paris JJ, Frye CA. Estrous cycle, pregnancy, and parity enhance performance of rats in object recognition or object placement tasks. Reproduction 2008;136:105–15. [8] Frye CA, Duffy CK, Walf AA. Estrogens and progestins enhance spatial learning of intact and ovariectomized rats in the object placement task. Neurobiol Learn Mem 2007;88:208–16. [9] Conrad CD, Jackson JL, Wieczorek L, Baran SE, Harman JS, Wright RL, et al. Acute stress impairs spatial memory in male but not female rats: influence of estrous cycle. Pharmacol Biochem Behav 2004;78:569–79. [10] Conrad CD, Galea LA, Kuroda Y, McEwen BS. Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment. Behav Neurosci 1996;110:1321–34. [11] Dellu F, Mayo W, Cherkaoui J, Le Moal M, Simon H. A two-trial memory task with automated recording: study in young and aged rats. Brain Res 1992;588:132–9. [12] Ennaceur A, Neave N, Aggleton JP. Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulate cortices, the medial prefrontal cortex, the cingulum bundle and the fornix. Exp Brain Res 1997;113:509–19.
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[47] Han X, Aenlle KK, Bean LA, Rani A, Semple-Rowland SL, Kumar A, et al. Role of estrogen receptor alpha and beta in preserving hippocampal function during aging. J Neurosci 2013;33:2671–83. [48] Luine VN. Sex steroids and cognitive function. J Neuroendocrinol 2008;20:866–72. [49] Dohanich GP, Fader AJ, Javorsky DJ. Estrogen and estrogen-progesterone treatments counteract the effect of scopolamine on reinforced T-maze alternation in female rats. Behav Neurosci 1994;108:988–92. [50] Fader AJ, Hendricson AW, Dohanich GP. Estrogen improves performance of reinforced T-maze alternation and prevents the amnestic effects of scopolamine administered systemically or intrahippocampally. Neurobiol Learn Mem 1998;69:225–40. [51] Marriott LK, Korol DL. Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning. Neurobiol Learn Mem 2003;80:315–22. [52] Lam TT, Leranth C. Role of the medial septum diagonal band of Broca cholinergic neurons in oestrogen-induced spine synapse formation on hippocampal CA1 pyramidal cells of female rats. Eur J Neurosci 2003;17:1997–2005.
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Research report
Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats Wayne R. Hawley a,∗ , Elin M. Grissom a , Nicole M. Moody b , Gary P. Dohanich a,b , Nandini Vasudevan b,c a
Department of Psychology, Tulane University, New Orleans, LA 70118, United States Program in Neuroscience, Tulane University, New Orleans, LA 70118, United States c Department of Cellular and Molecular Biology, Tulane University, New Orleans, LA 70118, United States b
h i g h l i g h t s • Estradiol treatment enhanced spatial recognition memory in ovariectomized rats. • G-1 treatment enhanced spatial recognition memory in ovariectomized rats. • Neither estradiol nor G-1 impacted levels of activity in ovariectomized rats.
a r t i c l e
i n f o
Article history: Received 17 November 2013 Received in revised form 4 January 2014 Accepted 7 January 2014 Available online 18 January 2014 Keywords: Estradiol Ovariectomy G-protein-coupled receptor 30 G-1 Spatial learning and memory
a b s t r a c t In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the ␣ or  isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 g) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 g) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Elevated levels of ovarian hormones impact performance on a variety of spatial learning and memory tasks [1–6], including those that take advantage of the tendency of rodents to seek out spatial novelty. These tasks include object location tasks [7,8] and a Ymaze task [9], two hippocampus-based cognitive tasks that are free
∗ Corresponding author at: Tulane University, Department of Psychology, 2007 Percival Stern Hall, New Orleans, LA 70118, United States. Tel.: +1 504 865 5331; fax: +1 504 862 8744. E-mail addresses: [email protected], [email protected] (W.R. Hawley). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2014.01.006
from the stress associated with water escape and the motivation to obtain food reward [10–12]. Paralleling the memory enhancement observed during proestrus, when levels of ovarian hormones are naturally elevated, ovariectomized rodents treated with estradiol also exhibited an enhancement in spatial learning and memory on a variety of tasks [13–18]. However, it is important to keep in mind that the effects of estradiol treatment vary as a function of strain, dose and behavioral testing paradigm [1]. For instance, treatment of ovariectomized rodents with lower doses of estradiol, which mimic the peak in physiological levels that characterize proestrus, tend to facilitate spatial learning and memory, whereas higher doses have been shown to impair performance [16,17,19–22]. As would be expected, the enhanced spatial learning and memory in ovariectomized rodents following estradiol treatment
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involves the classic estrogen receptor ␣ and  isoforms. For example, genetic deletion of estrogen receptor  abrogated the spatial memory enhancement that occurred in ovariectomized rodents treated with estradiol [23]. Alternatively, lentivirus mediated overexpression of estrogen receptor ␣ in the hippocampus [24,25], or administration of selective estrogen receptor ␣ or  modulators was sufficient to enhance spatial cognition in ovariectomized rats and mice [18,26–29]. In addition to the classic estrogen receptor isoforms, the memory enhancing effects of estradiol on spatial learning and memory are modulated by activation of the putative estrogen membrane receptors GPR30, which are located in the hippocampus, as well as other brain structures [30,31]. In ovariectomized rats, treatment with G-1, a specific agonist for estrogen membrane receptor GPR30, mimicked the enhancement in spatial learning that occurred on a delayed matching-to-position T-maze task following treatment with either estradiol, or selective agonists for estrogen receptor ␣ or  [27]. Alternatively, treatment with G-15, which is a GPR30 antagonist, abrogated the enhancing effects of estradiol treatment to ovariectomized rats on the same delayed matching-to-position T-maze task [32]. Therefore, because treatment of ovariectomized rats with G-1 mimicked the effects of estradiol treatment on a food-motivated spatial learning task [27], we predicted that G-1 treatment would enhance spatial recognition memory on a Y-maze task, an effect we expected to be paralleled following treatment with a low dose of estradiol.
2. Materials and methods 2.1. Animals All procedures, which were identical for both experiments, were approved by the Tulane University Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Female Long-Evans rats in Experiment 1 (N = 44) and Experiment 2 (N = 20) were purchased from Harlan Laboratories Inc. (Indianapolis, IN) and arrived at the animal care facility at Tulane University at approximately 60 days of age. Rats were housed in pairs, provided ad libitum access to food and water (Teklad Inc., Madison, WI), and maintained on a reversed 12:12 h light-dark cycle (lights off at 11:00 h) in animal care facilities at Tulane University accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Rats were acclimated to the vivarium for 5–7 days upon arrival to the facility before procedures began. In addition, rats were acclimated to procedural and behavioral testing rooms for at least 30 min prior to procedures or behavioral testing, which occurred during the dark phase of the light–dark cycle.
2.2. Ovariectomy At approximately 65 days of age, rats were ovariectomized under anesthesia induced by intraperitoneal injections of ketamine (100 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (7 mg/kg, Miles Laboratories, Shawnee, KS). Dorsal ventral incisions were made bilaterally approximately 5 cm from the most posterior point of the rib cage. After the ovaries were excised, ovarian blood vessels were ligated, the muscle wall was closed with 4.0 silk sutures (Teleflex Medical, Kenosha, WI), and the skin was closed with titanium wound clips (Mercer Glassware Inc., New York, NY), which were removed 10 days after surgery. Rats were provided free access to drinking water containing ibuprofen (25 mg/kg) to reduce post-surgical pain during their recovery for 3 consecutive days after surgery. Then, to acclimate to experimenters, rats were
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handled for 1 min each day for 7 consecutive days beginning 1 week after surgery. 2.3. Hormone (Experiment 1) and drug treatment (Experiment 2) On the last 2 days of handling, which occurred 48 and 24 h prior to beginning behavioral testing, rats in Experiment 1 were administered intramuscular injections of either sesame oil (0.1 ml; n = 10; Sigma–Aldrich Co., St. Louis, MO) or 17-estradiol benzoate (1 g, n = 12; 10 g, n = 10; or 25 g, n = 12; Sigma–Aldrich Co.) suspended in sesame oil (0.1 ml). Rats in Experiment 2 were administered intramuscular injections of either sesame oil (0.1 ml; Sigma–Aldrich Co.) or G-1 (25 g; Sigma–Aldrich Co.), the estrogen membrane receptor agonist, suspended in sesame oil (0.1 ml). The dose of G-1 was selected based on preliminary studies and findings which indicated that higher doses of specific estrogen receptor agonists are often necessary to mimic the effects of estradiol on spatial learning [18,28] and on the morphology of the hippocampus [17,29]. 2.4. Y-maze task Spatial recognition memory was characterized on a hippocampus-based version of a Y-maze task [10]. The maze was constructed from gray opaque Plexiglas that formed three identical arms (50 × 10 × 20 cm; Stoelting ANY-maze, Wood Dale, IL), and was surrounded by a variety of two and three dimensional extra-maze cues. To minimize the amount of time experimenters spent in proximity to the Y-maze, the start arm was placed in the testing environment in the position that provided the most efficient exit for experimenters. For both experiments, all rats were entered into the maze from the same start arm position within the testing environment. Rats were placed into the maze for a 15-min information trial and were allowed to freely explore the start arm and a second arm, but access to the third arm, which was counterbalanced between conditions, was blocked by an opaque plastic partition. After a delay period of 48 h, rats were placed back into the same start arm and allowed to freely explore all three arms for a 5-min retention trial, which was video recorded by and overhead camera for later scoring. The maze was cleaned thoroughly with 10% ethanol and air-dried after each trial to remove olfactory cues. Entry into an arm was defined as all four paws crossing into the arm proper by an experimenter blind to the conditions. On the retention trial, spatial recognition memory was indicated by calculating a novel arm preference index, which was the average of the percentage of entries and time into the arm associated with the novel environment relative to all of the arms. Greater preference for the arm associated with the novel environment indicated better spatial recognition memory. The total number of arms entered during the retention trial served as an indicator of general activity. However, because rats can quickly acclimate to novelty while exploring a Y-maze [33], spatial memory and activity were assessed for both the first minute, as well as the entire five minutes of the retention trial. 2.5. Statistical analyses Analyses of variance and independent samples t-tests were conducted to examine differences in activity between conditions on the retention trial in Experiment 1 and Experiment 2, respectively. One-sample t-tests (relative to chance, 33%) were conducted to determine within-group preference for the arm associated with the novel environment on the retention trial of the Y-maze task, which is an indicator of spatial recognition memory [34]. Statistical significance was indicated by p ≤ 0.05. Rats that failed to move from
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A Novel Arm Composite Score (%T+%E/2)
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Novel Arm Composite Score (%T+%E/2)
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44 33 22 11
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B Total Arm Entries
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18 16 14 12 10 8 6 4 2 0
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p = 0.06
Fig. 1. Effects of estradiol on activity and spatial recognition memory on the retention trial of a Y-maze task. (A) Only rats administered 1 g of estradiol 24 and 48 h prior to the information trial exhibited recognition of the novel arm during the first minute of the retention trial, as indicated by a preference for the novel arm relative to chance. (B) No differences in activity, as indicated by total arm entries, were found as a function of estradiol treatment. *p ≤ 0.05 relative to chance performance, 33%.
the start arm, as well as those that climbed on top of the walls of the Y-maze during either the information or retention trial were excluded from analyses. 3. Results 3.1. Experiment1: the effects of estradiol on spatial recognition memory As depicted in Fig. 1A, rats administered 1 g [t(6) = ± 2.39, p ≤ 0.05] of estradiol 48 and 24 h prior to the information trial of the Y-maze task, but not those administered oil [t(7) = ± 4.30, p ≤ 0.01], exhibited a preference for the arm associated with the novel environment during the first minute of the retention trial. Neither administration of 10 g [t(7) = ± 1.84, p = 0.11] nor 25 g [t(9) = ± 0.00, p = 1.00.] of estradiol increased preference for the arm associated with the novel environment during the first minute of the retention trial (Fig. 1A). Neither oil nor any of the doses of estradiol impacted preference for the arm associated with spatial novelty across the entire five minutes of the retention trial (Fig. 1A). Importantly, no differences in activity, as indicated by total arm entries on the retention trial, were found between rats administered oil and those administered estradiol (Fig. 1B). Therefore, as indicated during the early stage of the retention trial, a low dose of estradiol enhanced spatial recognition memory without impacting activity. 3.2. Experiment 2: the effects of the GPR30 agonist G1 on spatial recognition memory The results illustrated in Fig. 2A indicated that rats treated with G-1 prior to the information trial tended to exhibit a preference
Vehicle G-1 (25 µg)
18 16 14 12 10 8 6 4 2 0 1st min
All 5 min
All 5 min
All 5 min
Fig. 2. Effects of the estrogen membrane receptor agonist G-1 on activity and spatial recognition memory on the retention trial of a Y-maze task. (A) Only rats administered G-1 24 and 48 h prior to the information trial exhibited recognition of the novel arm, as indicated by a greater preference for novel arm relative to the familiar arm across the entire 5 min of the retention trial. (B) No differences in activity, as indicated by total arm entries, were found between rats administered oil or G-1. *p ≤ 0.05 relative to chance performance, 33%.
for the arm associated with the novel environment during the first minute of the retention trial [t(6) = ± 2.37, p = 0.06]. However, as the retention trial progressed, rats treated with G-1 exhibited a preference for the arm associated with the novel environment as indicated across the entire five minutes of the retention trial [t(6) = ± 6.61, p < 0.01]. Rats treated with oil did not exhibit a preference for the arm associated with the spatial novelty during the first minute [t(8) = ± 1.60, p = 0.15] or the entire five minutes [t(8) = ± 0.39, p = 0.71] of the retention trial (Fig. 2A). As depicted in Fig. 2B, no differences in activity, as indicated by total arm entries on the retention trial, were found between rats administered the estrogen membrane receptor agonist G-1 and those administered oil 24 and 48 h prior to the information trial. Therefore, in ovariectomized rats, administration of a GPR30 agonist was sufficient to enhance spatial recognition memory without impacting activity. 4. Discussion The GPR30 agonist, G1, enhanced spatial memory on the retention trial of a Y-maze task when administered to ovariectomized rats at 48 and 24 h before the information trial. Treatment with a low dose of estradiol at 48 and 24 h prior to the information trial of a Y-maze task similarly enhanced spatial recognition memory in ovariectomized rats on the retention trial conducted 48 h later. Because there were no differences in activity level, which was indicated by the total number of arm entries on the retention trial, it is reasonable to propose that the preference for the arm associated with spatial novelty that emerged as a function of estradiol treatment in Experiment 1, and administration of G-1 in Experiment 2, was not impacted by the non-mnemonic factor of activity.
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Collectively, the results of Experiments 1 and 2 indicated that the memory enhancing effects of estradiol in ovariectomized rats on a spatial task that takes advantage of the preference of rats to seek out novelty also occurs following treatment with G-1, a drug that selectively activates the putative estrogen membrane receptor GPR30.
4.1. Ovarian hormones and spatial cognition During proestrus, when ovarian hormones are naturally elevated, gonadally intact rats exhibit enhanced spatial cognition on a variety of tasks motivated by the preference of rats to gravitate toward novelty [7–9,35], an effect which also occurs in ovariectomized rodents following administration of estradiol [18]. Moreover, similar to both the low dose (1 g) and timing of estradiol treatment that enhanced memory for spatial novelty in Experiment 1, administration of estradiol (4.5 g/kg) at 48 and 24 h prior to training also has been shown to enhance learning on a foodmotivated version of a hippocampus-based place learning task [19]. Therefore, in ovariectomized rats, low dose regimens of estradiol treatment, which are designed to approximate the physiological levels that occur during proestrus, result in enhanced cognition on a variety of spatial learning and memory tasks. However, with this in mind, it is important to note that in the current study, levels of estradiol were not measured out of concern that the stress associated with blood sampling could have impacted the acquisition or consolidation of spatial information at the time of the information trial. In addition, examining levels of estradiol following completion of the retention trial, which would have been 72-h after the last injection, would have provided little information as to what levels were present during the information trial, when spatial information was being learned. Unlike tasks that rely upon detection of spatial novelty, the effects of ovarian hormones on both water maze tasks [2,4] and food-motivated radial-arm maze tasks [6,36] have been somewhat inconsistent, such that in some instances rats in proestrus exhibited poorer spatial learning and memory [3,37]. Arguably, the inconsistent results of ovarian hormones on water maze tasks may have been due to elevated levels of stress hormones [3,5], which have been shown to interfere with spatial cognition [38]. This interpretation is important to consider given the hyper-responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis during proestrus, an effect that has been attributed to estradiol [39]. Therefore, as may be the case for examining the effects of sex steroids on spatial cognition in male rats [34], behavioral tasks that are contingent upon the preference of rats to seek out novelty, and not by the stress associated with water escape, may be more suitable for examining the relationship between estradiol and spatial cognition in female rats. Neither estradiol nor G-1 treatment impacted the nonmnemonic factor of exploratory behavior, which was indicated by the total number of arm entries during the retention trial. Furthermore, across both experiments, only one rat, which was not included in the analyses, failed to move from the start arm during the information trial, which indicated that neither estradiol nor G-1 impacted the tendency to explore novelty. In accordance with these findings, systemic administration of G-1 has been shown not to impact anxiety-like behavior in ovariectomized rodents [40]. Despite these findings, as well as results that indicate greater preference for the novel arm of a Y-maze is an indicator of hippocampus-based spatial memory [10], we cannot entirely rule out the possibility that treatment with either estradiol or G-1 merely impacted the tendency to explore novelty during the retention trial, and not just memory for novelty.
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4.2. Estrogen receptors and spatial cognition In Experiment 2, activation of the putative estrogen membrane receptor GPR30 in ovariectomized rats following treatment with G-1 resulted in an enhancement in spatial recognition memory. Following administration of estradiol conjugated to cyclodextrin, which activates estrogen-associated membrane receptors, ovariectomized rodents exhibited enhanced performance on a spatial version of the water maze task [41] and on object memory tasks, which characterize spatial and non-spatial memory [42,43]. However, only two other studies, both from the same laboratory, have directly examined the relationship between GPR30 and spatial cognition in rats [27,32]. Treatment of ovariectomized rats with G-1 for over 1 month resulted in enhanced spatial learning on a foodmotivated version of a delayed matching-to-position T-maze task, an effect that mimicked the beneficial effects of estradiol treatment [27]. In a follow-up study, treatment of ovariectomized rats for over 1 month with a GPR30 antagonist abrogated the enhancing effects of simultaneous estradiol treatment on the same spatial task [32]. Therefore, the results from Experiment 2 indicated that short-term treatment with G-1, which mimicked the short-term treatments with estradiol used in Experiment 1 and elsewhere [19,22], was sufficient to enhance spatial cognition in ovariectomized rats on a task not motivated by food reward. Although the results of Experiment 2 illustrate a role for GPR30 in spatial recognition memory, the contributions of estrogen receptors ␣ and  to spatial recognition memory remain to be determined. However, administration of agonists that selectively activate either estrogen receptor ␣ or  have been show to enhance performance on object placement tasks, which also rely on detection of spatial novelty [8,18,23]. Furthermore, when levels of estradiol are elevated, expression of estrogen receptor  was necessary for enhancements in spatial cognition to occur on an object location task [23] and a spontaneous spatial alternation task [35]. Therefore, in addition to GPR30, both the estrogen receptor ␣ and  isoforms impact learning and memory on tasks that rely upon the detection of spatial novelty. 4.3. Possible mechanisms mediating effects of estradiol and G-1 on spatial memory The memory enhancing effects of estradiol in Experiment 1 emerged during the first minute of the retention trial, whereas the beneficial effects of G-1 in Experiment 2 were observed across the entire 5-min of the retention trial. As suggested by Wright and colleagues [33], rats can quickly acclimate to a Y-maze, which may explain why rats treated with estradiol in Experiment 1 exhibited a preference for the novel arm only during the first minute. However, it also is important to consider how the distribution of specific estrogen receptor subtypes within the hippocampus, a brain structure that modulates spatial recognition memory [10], may have impacted the time course in novel arm preference as a function of estradiol and G-1 treatment. GPR30 is seemingly expressed to a greater extent in the rat hippocampus than estrogen receptor ␣ and  [30,44,45]. Interestingly, estrogen receptor  has been shown to repress transcriptional processes induced by estrogen receptor ␣ [46]. Although the interactive nature of GPR30 and estrogen receptors ␣ and  remains to be determined, interactions between estrogen receptor ␣ and  isoforms can subsequently impact the influence of estradiol on spatial cognition [47]. Conceivably, in the current study, treatment with estradiol, which activates all estrogen receptor subtypes, may have altered the beneficial effects of GPR30 activation. The reported effects of gonadal steroids, including estradiol, on spatial cognition are typically modest in unmanipulated rodents [1,48]. Prominent effects of estradiol emerge when performance
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has been compromised, for example, when cholinergic function is impaired by administration of agents designed to mimic the pathology associated with Alzheimer’s disease [49,50]. In addition to the hippocampus, GPR30 also is expressed in the basal forebrain, a brain region where administration of G-1 has been shown to mimic the stimulatory effects of estradiol on potassium-stimulated acetylcholine release within the hippocampus [31]. Interestingly, short-term treatment of ovariectomized rats with estradiol, which mimicked the time-course of estradiol treatment in Experiment 1 and G-1 administration in Experiment 2, elevated levels of acetylcholine release in the hippocampus during learning on a spatial task [51]. Furthermore, in ovariectomized rats, estradiol-induced up-regulation of synaptic spines in the hippocampus was contingent upon cholinergic innervation from the basal forebrain [52]. Given the increase in dendritic spine densities that occurs in the hippocampus of ovariectomized rodents following a single treatment with even a very low dose of estradiol [17], it is reasonable to believe that in addition to greater acetylcholine release [51] there also was an increase in synaptic connectivity during learning in rats treated with either estradiol or G-1. Arguably, the spatial recognition memory enhancement that occurred in rats treated with either estradiol or G-1 was mediated by activation of GPR30 in the basal forebrain, such that activation of GPR30 in the basal forebrain elevated levels of acetylcholine release in the hippocampus [31], which resulted in increased synaptic connectivity and enhanced neural communication during learning. Role of funding sources This research was supported by a National Foundation of Science CAREER award (#1053716) and Flowerree Summer Fellowships. Acknowledgement The authors gratefully acknowledge the expert supervision of animal care provided by Kimberly Scamardo. References [1] Dohanich G. Gonadal steroids, learning, and memory. In: Pfaff D, Arnold A, Etgen A, Fahrback S, Rubin R, editors. Hormones, brain and behavior. San Diego: Academic Press; 2002. p. 265–327. [2] Berry B, McMahan R, Gallagher M. Spatial learning and memory at defined points of the estrous cycle: effects on performance of a hippocampaldependent task. Behav Neurosci 1997;111:267–74. [3] Warren SG, Juraska JM. Spatial and nonspatial learning across the rat estrous cycle. Behav Neurosci 1997;111:259–66. [4] Frick KM, Berger-Sweeney J. Spatial reference memory and neocortical neurochemistry vary with the estrous cycle in C57BL/6 mice. Behav Neurosci 2001;115:229–37. [5] Rubinow MJ, Arseneau LM, Beverly JL, Juraska JM. Effect of the estrous cycle on water maze acquisition depends on the temperature of the water. Behav Neurosci 2004;118:863–8. [6] Pompili A, Tomaz C, Arnone B, Tavares MC, Gasbarri A. Working and reference memory across the estrous cycle of rat: a long-term study in gonadally intact females. Behav Brain Res 2010;213:10–8. [7] Paris JJ, Frye CA. Estrous cycle, pregnancy, and parity enhance performance of rats in object recognition or object placement tasks. Reproduction 2008;136:105–15. [8] Frye CA, Duffy CK, Walf AA. Estrogens and progestins enhance spatial learning of intact and ovariectomized rats in the object placement task. Neurobiol Learn Mem 2007;88:208–16. [9] Conrad CD, Jackson JL, Wieczorek L, Baran SE, Harman JS, Wright RL, et al. Acute stress impairs spatial memory in male but not female rats: influence of estrous cycle. Pharmacol Biochem Behav 2004;78:569–79. [10] Conrad CD, Galea LA, Kuroda Y, McEwen BS. Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment. Behav Neurosci 1996;110:1321–34. [11] Dellu F, Mayo W, Cherkaoui J, Le Moal M, Simon H. A two-trial memory task with automated recording: study in young and aged rats. Brain Res 1992;588:132–9. [12] Ennaceur A, Neave N, Aggleton JP. Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulate cortices, the medial prefrontal cortex, the cingulum bundle and the fornix. Exp Brain Res 1997;113:509–19.
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