Intra-perirhinal cortex administration of estradiol, but not an ERβ agonist, modulates object-recognition memory in ovariectomized rats
Introduction
Object-recognition memory (ORM), which is the ability to discriminate the familiarity of objects previously encountered (Aggleton & Brown, 1999), is modulated by 17β-estradiol (E2) in humans (Craig et al., 2010), nonhuman primates (Lacreuse et al., 2000, Rapp et al., 2003, Voytko et al., 2008) and rodents (Luine, 2015). In humans and nonhuman primates, this ability is typically assessed using either the delayed nonmatching-to-sample (DNMS), or delayed matching-to-sample (DMS) tasks. Both tasks involve two phases; the Familiarization phase, when one object is presented, and after a delay, the Test phase is given and involves presentation of the same object (or a copy) along with a novel object. When nonhuman primates are used, a food reward is given when the correct object is displaced (novel object for the DNMS task, and sample object for the DMS task), and so both tasks are appetitively motivated and involve making an explicit choice response on each trial. A different set of objects are given on each trial, and once reliable performance is achieved, longer delays (ranging from several seconds to minutes) between each phase can be introduced to increase memory load. The strength of ORM is assessed by calculating the percentage of trials within a test session in which a correct choice is made (i.e. accuracy scores). Surgical or pharmacological ovariectomy (OVX) is associated with lower accuracy relative to intact (Craig et al., 2010, Lacreuse et al., 2000) or OVX with E2 replacement (Rapp et al., 2003, dose: 100 μg/ml, i.m. every 3 weeks; Voytko et al., 2008, dose: acute: 0.1–0.2 mg/ml, i.m. every 12 days, and chronic: 40–80 pg/ml via silastic implants), although one study reported no effect (Lacreuse & Herndon, 2003).
ORM in rodents is generally evaluated using the Novel-Object Preference (NOP) test (also referred to as the Novel-Object Recognition task, the Object Recognition Memory task, and the Spontaneous Object Recognition task). Similar to the D(N)MS tasks, the NOP test is divided into two phases. During the Familiarization phase, rodents can explore two identical copies of an object that later serves as the sample object. Following a retention delay, the Test phase is given, when a third copy of the sample object is presented along with a novel object. Rats will typically spend more time with the novel relative to the familiar object following retention delays lasting several minutes to weeks (Ennaceur and Aggleton, 1994, Ennaceur and Delacour, 1988, Gaskin et al., 2003, Mumby et al., 2007, Mumby et al., 2005). When this occurs, intact ORM can be inferred, as the subject must be able to detect the familiarity of the sample in order to demonstrate a preference for the novel object. Normal performance on either the NOP or D(N)MS tests requires ORM, but the two tests differ in terms of other performance requirements and the outcome measures used to draw conclusions about treatment effects on ORM. As described above, explicit choice responses are used for the D(N)MS tasks to infer the strength of ORM, with higher accuracy reflecting better ORM ability. The NOP test does not require an explicit choice from the subject. Instead, spontaneous object investigation is recorded, and intact ORM is assumed when more time is spent with the novel object. This spontaneous preference for the novel object is referred to as novelty preference in the present paper. Novelty preference is differentiated from ORM in that it refers to the outcome measure of this test, while ORM refers to the ability to recognize the sample object.
Studies report impaired novelty preference following OVX or chronic low E2 replacement (∼22 pg/ml serum via silastic implants; Gervais, Jacob, Brake, & Mumby, 2013) relative to moderate to high E replacement (rats: 5–30 μg/kg acute, s.c., Gervais et al., 2013, Inagaki et al., 2010, Luine et al., 2003; mice: 1.5 μg/kg −0.4 mg/kg, acute, s.c., Gresack and Frick, 2006, Phan et al., 2012; 1–2.5 μM, chronic, p.o., Fernandez & Frick, 2004), or gonadally-intact females (Wallace, Luine, Arellanos, & Frankfurt, 2006). These studies support the idea that E2 enhances novelty preference in rodents following retention delays ranging from 5 min to 72 h (for a review, see Luine, 2015).
The majority of studies examining local effects of E2 on ORM have focussed on the hippocampus (HPC). Specifically, these studies report enhanced novelty preference following intra-HPC infusion of E2 (Boulware et al., 2013, Fernandez et al., 2008, Lewis et al., 2008, Phan et al., 2012), and many have concluded that E2 enhances ORM via actions within the HPC. However, ORM is dependent upon the functioning of the perirhinal cortex, as damage to this structure results in impaired performance on both the D(N)MS tasks and NOP test (PRh; Warburton and Brown, 2014, Winters et al., 2008). Despite this, only one study to date has examined the local effects of E2 in this region. Gervais et al. (2013) report that local infusions of E2 (244.8 pg/μl) in the PRh and entorhinal cortex (EC) enhance novelty preference on a 72-h retention test (Gervais et al., 2013) in OVX rats given chronic low E2 replacement (∼22 pg/ml serum). Recently, E2 has also been shown to reduce the density of mushroom-shaped (i.e. mature) spines in Broadmann’s area 35 of the PRh (Gervais, Mumby, & Brake, 2015). Although preliminary, these data suggest that E2 may modulate ORM by altering the spine density in the PRh.
Estrogen receptors (ERs) are thought play a role in activating intracellular signaling molecules that promote both spine plasticity and learning and memory (Gabor, Lymer, Phan, & Choleris, 2015). All three estrogen receptors (ERα, ERβ, GPER1) are expressed in the PRh (Blurton-Jones and Tuszynski, 2002, Brailoiu et al., 2007, Hazell et al., 2009, Shughrue et al., 1997, Shughrue and Merchenthaler, 2001). However, it is currently unknown whether these receptors in the PRh play a role in ORM. There is evidence that ERβ is highly expressed in the PRh (Shughrue et al., 1997), and systemic administration of ERβ agonists influence novelty preference (Jacome et al., 2010, Walf et al., 2008, Walf et al., 2009). For example, acute administration of diarylpropionitrile (DPN, 3 mg/kg, s.c.), a selective ERβ agonist, to OVX rats results in novelty preference whereas administration of propyl pyrazole triol (PPT, 3–5 mg/kg, s.c.), a selective ERα agonist, or vehicle does not (Jacome et al., 2010). Therefore, one aim of the present study was to determine whether intra-PRh infusion of an ERβ agonist influences performance on ORM tests.
There are reasons for scepticism regarding the conclusion that enhanced novelty preference following infusion of E2 reflects improved ORM abilities. Recent evidence raises concern about the internal validity of the NOP test as a method of indexing ORM. For example, there is no relationship between the amount of time a subject spends investigating objects during the familiarization phase and the magnitude of novelty preference (Gaskin et al., 2010, Gervais et al., 2013). Also, prolonged or repeated exposure to a sample object does not change the magnitude of novelty preference (Gaskin et al., 2010), although both manipulations would undoubtedly lead to stronger memory of the object. Together, these results suggest that stronger novelty preference does not necessarily indicate superior ORM abilities. Perhaps the most important issue with the NOP test concerns understanding what it means about ORM when a rodent fails to demonstrate a preference for the novel object. Although demonstrating a preference for the novel object requires recognizing the sample object, rodents can recognize the sample object and still fail to demonstrate a preference. There are even instances when rodents display a preference for the sample object during the Test phase (Mumby et al., 2002, Vargas-López et al., 2015). Preference for either the sample or novel object requires intact ORM, but only novelty preference is typically associated with this ability. Taken together, these findings question conclusions that are made based on studies that use the NOP test as the sole measure of ORM.
In light of the concerns described above, one study (Gervais et al., 2013) used both the NOP test and DNMS task to assess ORM and divergent effects on both tests were reported. Ovariectomized rats received either systemic chronic low E2 (∼22 pg/ml serum) replacement alone or in combination with systemic acute high estradiol replacement (EB; 10 μg). In a separate experiment, the same rats received intracranial infusions of E2 (244.8 pg/μl) or vehicle into the PRh/EC. A within-subjects design was used, so each rat was tested under all hormonal conditions on both tests and at all retention delays. While acute high E replacement and intra-PRh/EC E2 were both associated with enhanced novelty preference following a 72-h retention delay on the NOP test, they were also associated with reduced accuracy on the DNMS task. Under both chronic low E2 replacement, and intra-PRh/EC infusions of vehicle, rats failed to demonstrate novelty preference on the NOP test, but performed better on the DNMS task than the acute high E replacement/E2-infuson condition. Since it is unlikely that ORM was both impaired and improved under the same hormone condition in the same set of animals, it seems reasonable that these rats continued to recognize the sample object on the NOP test (as they did on the DNMS task), but failed to show an exploratory preference. These findings suggest that while E2 in the PRh/EC can enhance novelty preference, this effect is probably not due to an improvement in ORM.
Given the divergent effects observed by Gervais et al. (2013), ORM in the present study was assessed using both accuracy on the DNMS task and novelty preference. Using a within-subjects design, OVX rats under chronic low E2 replacement (∼22 pg/ml serum) received intra-PRh infusions of DPN, E2, or vehicle prior to NOP test trials and DNMS test sessions. Two retention delays (4- & 72-h) and 4 retention delays (0.5, 2, 3, and 5 min) were used on the NOP test and DNMS task respectively. Previous studies demonstrate that E2 enhances novelty preference when administered either immediately before Familiarization, or immediately after, but not 2–3 h later (for a review, see Tuscher, Fortress, Kim, & Frick, 2015). Since the DNMS task is restricted to very brief delays, it is only feasible for infusions to occur prior to each test session, and so all infusions were given in this manner for both tests. The two retention delays used for the NOP test were chosen as they reflect the shortest and longest retention delay used previously in studies examining the role of E2 in novelty preference (Gervais et al., 2013, Inagaki et al., 2010). The retention delays used on the DNMS task are the same delays used by Gervais et al. (2013). To address whether the divergent results reported by Gervais et al. (2013) reflect a dissociation in E2′s effect on short- and long-term memory, object investigation was scored on both tests, allowing for the assessment of novelty preference across retention delays varying from 0.5-min to 72-h. Performance on each test was compared across the same three infusion conditions.
Section snippets
Subjects
Thirty-six Long Evans female rats (DNMS task: N = 24, 240–440 g; NOP test: N = 12, 260–430 g) bred in-house served as subjects. Rats were housed in pairs in transparent shoebox cages lined with a combination of woodchip and corncob bedding under 12:12 reverse light cycle (lights on at 8:00 pm) with ad libitum access to water and 30–40 g daily access to chow. Restricted access to food was provided as it is associated with improved health, longevity and reduced variability in body weight compared to ad
Histology
Following all behavioral testing, rats received a lethal dose of sodium pentobarbital followed by transcardial perfusions with 0.9% saline (250 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH = 7.4, 250 ml). The brains were excised and stored in 4% paraformaldehyde solution for 4 h before being transferred to a 30% sucrose/water solution overnight. The next day, the brains were transferred to a −80 °C freezer until sectioning. Using a cryostat microtome, 40 μm coronal
Discussion
The divergent results on the DNMS task and NOP test following intra-PRh/EC infusions of E2 reported by Gervais et al. (2013) were replicated in the present study. Intra-PRh infusions of E2 (244.8 pg/μl) were associated with lower accuracy on the DNMS task following a 5-min retention delay relative to when the vehicle was infused. In our previous study, reduced accuracy following infusions of E2 were observed following a 3-min retention delay. The effect size estimates (present study: Hedge’s g =
Acknowledgements
We are grateful to Jordan O’Byrne, Andrea Di Carlo, Meagan-Barrett-Bernstein, Sasha Van Frank Adler, Maria Jose Luna Chacon, Thiffya Arabi Kugathasan, Sophie Schmied, Jordyn Hanover, and Ayman Elsamman for their help with data collection. We are thankful to Aileen Murray and the Animal Care Facility staff for helping in the long-term care of the animals.
This work was supported by the National Science and Engineering Research Council of Canada Discovery Grants to DGM (156937-212). NJG was the
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