Spatial working memory in the touchscreen operant platform is disrupted in female rats by ovariectomy but not estrous cycle

https://doi.org/10.1016/j.nlm.2017.07.010Get rights and content

Highlights

  • Estrous cycle does not significantly affect performance in the touchscreen TUNL task.

  • Ovariectomy reduces spatial working memory at larger spatial separations.

  • Ovariectomised rats shift cognitive strategy when tested using a novel spatial separation.

Abstract

Learning and memory deficits have been described in rats and mice after ovariectomy (OVX) and across the estrous cycle. Preclinical researchers therefore often avoid using female animals and, consequently, a large male bias exists in the preclinical cognitive literature. In the present study we examined the role of sex hormones in the touchscreen operant platform using the spatial working memory trial unique nonmatching-to-location (TUNL) task.

Twenty-nine Long Evans rats were trained to acquire the TUNL task including three incremental spatial separations (S0, S1, S2). Following 20 consecutive days of training, subjects in experiment 1 (n = 15) remained intact and immediately progressed to TUNL testing, while subjects in experiment 2 were OVX (n = 6) or sham-operated (n = 8) prior to testing. Subjects were tested on 4 spatial separations (S0-3) with a 1 s or 6 s delay between the sample and nonmatching stimuli. The estrous cycle of intact rats was monitored during the 4 weeks of testing.

The estrous cycle phase did not significantly affect performance. In contrast, compared to intact rats, OVX impaired performance at larger spatial separations (S2-3) during the 1 s delay condition. Further, during the 6 s delay, OVX impaired S2 performance, however not S3. Our results suggest a probable shift in cognitive strategy following OVX, when tested with a large and novel spatial separation. Our findings suggest that ovarian hormone deprivation following OVX, but not estrous cycle, impairs spatial working memory as measured by the TUNL task. This research is relevant for future studies utilising the touchscreen TUNL task and for cognitive testing of female rats.

Introduction

It is well established that sex hormones, particularly estrogen, can affect cognitive performance (Luine, 2014, Sbisa et al., 2017). For example, in preclinical research, removal of the primary source of sex hormones via ovariectomy (OVX) can induce deficits in hippocampal-mediated spatial working memory and recognition memory (Inagaki et al., 2010, Luine et al., 1998). Treatment with exogenous estrogen, more specifically 17β-estradiol, can reverse cognitive impairments induced by OVX (Frye et al., 2007, Luine et al., 2003, Luine et al., 1998). It is worth noting, however, that estrogen treatment can have opposing effects depending on the cognitive domain tested, and the dose and duration of treatment (Wide, Hanratty, Ting, & Galea, 2004). For example, a high dose of chronic estrogen can impair non-spatial working memory in OVX rats, however a low dose can facilitate performance (Wide et al., 2004). Similarly, Inagaki et al. (2010) demonstrate a low dose of acute estrogen facilitates OVX rat non-spatial memory, while an intermediate dose of estrogen facilitates spatial memory (Inagaki et al., 2010).

The 4–5 day rat estrous cycle has also been found to influence performance in certain cognitive tasks (Abbott et al., 2016, Frye, 1995), however research thus far is inconsistent. For example, during proestrus, when estrogen and progesterone levels peak, performance in the Morris water maze is decreased, however only during task acquisition (Frye, 1995). In contrast, Warren and Juraska (1997) found a cued task in the Morris water maze was enhanced during proestrus when compared to estrus, a phase of lower estrogen levels. Pompili, Tomaz, Arnone, Tavares, and Gasbarri (2010) found no differences across the cycle in rats in the radial-arm maze using data from 20 consecutive estrous cycles. Similarly, no alterations in acquisition or performance have been seen across the estrous cycle in the delayed nonmatching-to-sample radial-arm maze task (Stackman, Blasberg, Langan, & Clark, 1997). Thus it is important to note that, while it is well substantiated that gonadal hormones can affect cognitive performance, research outcomes largely depend on the cognitive assay (Luine, 2014).

Automated touchscreen operant chambers are increasingly used for testing cognition in rats and mice. Compared to traditional measures, the platform involves minimal stress, very little experimenter involvement, and the ability to use cognitive tasks similar to those used in clinical studies (Bussey et al., 2012, Oomen et al., 2013). There are surprisingly few published studies to date on touchscreen cognitive testing in female rats and mice (Bussey et al., 2008). Indeed, the Nature Protocol for the touchscreen platform notes using male rodents has the advantage of avoiding potential estrous cycle-related variability in performance (Oomen et al., 2013), however, the effect of estrous cycle is yet to be measured in the touchscreen apparatus. Of note, in one study, male and female rats performed equally on the visual discrimination and reversal learning touchscreen task (Bussey et al., 2008).

The trial-unique nonmatching-to-location (TUNL) touchscreen task is a measure of spatial working memory, similar to the traditional delayed nonmatching-to-position task (Talpos, McTighe, Dias, Saksida, & Bussey, 2010). The TUNL task, which offers numerous locations for the sample and nonmatching square, is considered to reduce predictability and the possibility of mediating strategies (Oomen et al., 2013), and allows for flexibility in protocol, for example by modifying spatial distance or altering the delay period. Both the spatial and working memory components of the TUNL task are sensitive to hippocampal lesions (Talpos et al., 2010), while medial prefrontal cortical lesions impair working memory, however not spatial separation, in the TUNL task (McAllister, Saksida, & Bussey, 2013). The necessity for hippocampal integrity (Talpos et al., 2010) suggests the TUNL task is likely sensitive to removal of gonadal hormones, and fluctuations in endogenous estrogens and progestins during the estrous cycle (Foy, Baudry, Brinton, & Thompson, 2008). Previous research has demonstrated hippocampal dendritic spine and synapse density increases during proestrus, and declines by more than 30% during the low estrogen phase of estrus (Gould et al., 1990, Woolley and McEwen, 1992). Further, a similar decrease in hippocampal dendritic cell spine density is seen following OVX, while estrogen treatment can reverse this reduction (Woolley & McEwen, 1993).

Given the paucity of studies investigating cognitive performance of female rats in touchscreen tasks, the current study aimed to explore variability in performance due to the estrous cycle in intact female rats, and examine TUNL performance following removal of the gonadal hormones via OVX. Considering previous research using traditional cognitive measures, we hypothesise variable performance in spatial working memory across phases of the estrous cycle, and reduced performance following OVX.

Section snippets

Animals

Twenty-nine female Long Evans rats (experiment 1n = 15, experiment 2n = 14) were used in this study (Florey Institute of Neuroscience and Mental Health, Victoria, Australia). Animals were housed at the La Trobe University Animal Research and Teaching Facility in groups of 2–4 in individually-ventilated cages (Tecniplast Greenline Double Decker IVC), and were given one week to acclimate to the facility prior to behavioural testing. Rats were food restricted to 85–95% of their free-feeding weight

TUNL training

In intact female rats, TUNL training performance was analysed using a repeated measures ANOVA with separation (S0-2) and block (4 blocks × 5 consecutive days) as repeated factors. As expected, there was a significant difference in %correct between the four blocks, (F(3, 42) = 8.39, p  0.001; Supplementary Fig. 4), which included all spatial separations (S0-2). Bonferroni-adjusted pairwise comparisons showed block 2 (p = 0.010), and block 4 (p = 0.003) were significantly different from block 1, reflecting

Discussion

The current study aimed to investigate spatial working memory in female rats in the touchscreen TUNL task following OVX, and explore variability in performance due to the estrous cycle in intact rats. Consistent with previous literature using traditional cognitive assays (Cao et al., 2013, Ibrahim et al., 2016, Uzum et al., 2016, Wilson et al., 1999), removal of gonadal hormones via OVX induced cognitive impairment and a significant reduction in performance was observed at larger spatial

Acknowledgements

The authors are grateful to Dr Amy Reichelt for critically reading the manuscript. The authors also gratefully acknowledge the financial support of the National Health and Medical Research Council of Australia and the Jared Purton Foundation. The Florey Institute of Neuroscience and Mental Health acknowledges the support from the Victorian Government’s Operational Infrastructure Support Grant.

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