Trial-Unique Non-matching To Location Research Articles

Cognitive training of mice attenuates age-related decline in associative learning and behavioral flexibility.

Identifying factors that influence age-related cognitive decline is crucial, given its severe personal and societal impacts. However, studying aging in human or animal models is challenging due to the significant variability in aging processes among individuals. Additionally, longitudinal and cross-sectional studies often produce differing results. In this context, home-cage-based behavioral analysis over lifespans has emerged as a significant method in recent years. This study aimed to explore how prior experience affects cognitive performance in mice of various age groups (4, 12, and 22 months) using a home-cage-based touchscreen test battery. In this automated system, group-housed, ID-chipped mice primarily obtain their food during task performance throughout the day, motivated by their own initiative, without being subjected to food deprivation. Spatial working memory and attention were evaluated using the trial unique non-matching to location (TUNL) and the five-choice serial reaction time task (5-CSRTT), respectively. The same set of mice learned both of these demanding tasks. While signs of cognitive decline were already apparent in middle-aged mice, older mice exhibited poorer performance in both tasks. Mice at both 12 and 22 months displayed an increase in perseverance and a decrease in the percentage of correct responses in the TUNL test compared to the 4-month-old mice. Furthermore, during the 5-CSRTT, they exhibited higher rates of omissions and premature responses compared to their younger counterparts. Additionally, the correct response rate in 22-month-old mice was lower than that of the 4-month-old ones. However, mice that had undergone cognitive training at 4 months maintained high-performance levels when re-tested at 12 months, showing an increase in correct responses during TUNL testing compared to their untrained controls. In the 5-CSRTT, previously trained mice demonstrated higher correct response rates, fewer omissions, and reduced premature responses compared to naive control mice. Notably, even when assessed on a visual discrimination and behavioral flexibility task at 22 months, experienced mice outperformed naive 4-month-old mice. These findings highlight the advantages of early-life cognitive training and suggest that its benefits extend beyond the cognitive domains primarily targeted during early training. The success of this study was significantly aided by the fully automated home-cage-based testing system, which allows for high throughput with minimal human intervention.

Perineuronal nets are associated with decision making under conditions of uncertainty in female but not male mice

Biological sex influences decision-making processes in significant ways, differentiating the responses animals choose when faced with a range of stimuli. The neurobiological underpinnings that dictate sex differences in decision-making tasks remains an important open question, yet single-sex studies of males form most studies in behavioural neuroscience. Here we used female and male BALB/c mice on two spatial learning and memory tasks and examined the expression of perineuronal nets (PNNs) and parvalbumin interneurons (PV) in regions correlated with spatial memory. Mice underwent the aversive active place avoidance (APA) task or the appetitive trial-unique nonmatching-to-location (TUNL) touchscreen task. Mice in the APA cohort learnt to avoid the foot-shock and no differences were observed on key measures of the task nor in the number and intensity of PNNs and PV. On the delay but not separation manipulation in the TUNL task, females received more incorrect trials and less correct trials compared to males. Furthermore, females in this cohort exhibited higher intensity PNNs and PV cells in the agranular and granular retrosplenial cortex, compared to males. These data show that female and male mice perform similarly on spatial learning tasks. However, sex differences in neural circuitry may underly differences in making decisions under conditions of uncertainty on an appetitive task. These data emphasise the importance of using mice of both sexes in studies of decision-making neuroscience.

Mouse Behavior on the Trial-Unique Nonmatching-to-Location (TUNL) Touchscreen Task Reflects a Mixture of Distinct Working Memory Codes and Response Biases.

The trial-unique nonmatching to location (TUNL) touchscreen task shows promise as a translational assay of working memory (WM) deficits in rodent models of autism, ADHD, and schizophrenia. However, the low-level neurocognitive processes that drive behavior in the TUNL task have not been fully elucidated. In particular, it is commonly assumed that the TUNL task predominantly measures spatial WM dependent on hippocampal pattern separation, but this proposition has not previously been tested. In this project, we tested this question using computational modeling of behavior from male and female mice performing the TUNL task (N = 163 across three datasets; 158,843 trials). Using this approach, we empirically tested whether TUNL behavior solely measured retrospective WM, or whether it was possible to deconstruct behavior into additional neurocognitive subprocesses. Overall, contrary to common assumptions, modeling analyses revealed that behavior on the TUNL task did not primarily reflect retrospective spatial WM. Instead, behavior was best explained as a mixture of response strategies, including both retrospective WM (remembering the spatial location of a previous stimulus) and prospective WM (remembering an anticipated future behavioral response) as well as animal-specific response biases. These results suggest that retrospective spatial WM is just one of a number of cognitive subprocesses that contribute to choice behavior on the TUNL task. We suggest that findings can be understood within a resource-rational framework, and use computational model simulations to propose several task-design principles that we predict will maximize spatial WM and minimize alternative behavioral strategies in the TUNL task.SIGNIFICANCE STATEMENT Touchscreen tasks represent a paradigm shift for assessment of cognition in nonhuman animals by automating large-scale behavioral data collection. Their main relevance, however, depends on the assumption of functional equivalence to cognitive domains in humans. The trial-unique, delayed nonmatching to location (TUNL) touchscreen task has revolutionized the study of rodent spatial working memory. However, its assumption of functional equivalence to human spatial working memory is untested. We leveraged previously untapped single-trial TUNL data to uncover a novel set of hierarchically ordered cognitive processes that underlie mouse behavior on this task. The strategies used demonstrate multiple cognitive approaches to a single behavioral outcome and the requirement for more precise task design and sophisticated data analysis in interpreting rodent spatial working memory.

A Preclinical Model of Computerized Cognitive Training: Touchscreen Cognitive Testing Enhances Cognition and Hippocampal Cellular Plasticity in Wildtype and Alzheimer's Disease Mice.

With the growing popularity of touchscreen cognitive testing in rodents, it is imperative to understand the fundamental effects exposure to this paradigm can have on the animals involved. In this study, we set out to assess hippocampal-dependant learning in the APP/PS1 mouse model of Alzheimer’s disease (AD) on two highly translatable touchscreen tasks – the Paired Associate Learning (PAL) task and the Trial Unique Non-Matching to Location (TUNL) task. Both of these tests are based on human tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and are sensitive to deficits in both mild cognitive impairment (MCI) and AD. Mice were assessed for deficits in PAL at 9–12 months of age, then on TUNL at 8–11 and 13–16 months. No cognitive deficits were evident in APP/PS1 mice at any age, contrary to previous reports using maze-based learning and memory tasks. We hypothesized that daily and long-term touchscreen training may have inadvertently acted as a cognitive enhancer. When touchscreen-tested mice were assessed on the Morris water maze, they showed improved task acquisition compared to naïve APP/PS1 mice and wild-type (WT) littermate controls. In addition, we show that touchscreen-trained WT and APP/PS1 mice show increased cell proliferation and immature neuron numbers in the dentate gyrus compared to behaviorally naïve WT and APP/PS1 mice. This result indicates that the touchscreen testing paradigm could improve cognitive performance, and/or mask an impairment, in experimental mouse models. This touchscreen-induced cognitive enhancement may involve increased neurogenesis, and possibly other forms of cellular plasticity. This is the first study to show increased numbers of proliferating cells and immature neurons in the hippocampus following touchscreen testing, and that touchscreen training can improve cognitive performance in maze-based spatial navigation tasks. This potential for touchscreen testing to induce cognitive enhancement, or other phenotypic shifts, in preclinical models should be considered in study design. Furthermore, touchscreen-mediated cognitive enhancement could have therapeutic implications for cognitive disorders.

Capturing longitudinal impacts on cognition following stroke in rodent models using touchscreen testing

AbstractBackgroundUp to two thirds of ischaemic stroke survivors develop cognitive impairment, predominantly impacting executive and attentional processing. Animal models are essential for effective translation and development of therapies. However, accurately modelling cognitive dysfunction in animal models of stroke has been limited. To address this, we aimed to longitudinally examine changes in attentional processing, spatial working memory and processing speed using two rodent ischemic injury models: the middle cerebral artery occlusion (MCAO) in C57BL/6J mice and the endothelin‐1 injury in Long Evans rats.MethodUsing the rodent touchscreen cognitive platform, we assessed attention in mice and rats employing the Continuous Performance Task (CPT) paradigm, and spatial working memory in mice using the trial unique non‐matching to location (TUNL) paradigm. Cognitive performance in adult mice and rats was assessed before inducing stroke, and at 4, 12, 24, 36 and 48 weeks post‐injury.ResultMice exposed to MCAO showed robust, long‐lasting deficits in attentional processing and spatial working memory post‐stroke. In comparison, we observed no changes in attentional processing following endothelin‐1 injury to the motor cortex in rats.ConclusionOur findings provide the first evidence for longitudinal cognitive changes in a rodent model of stroke, recapitulating that observed in the clinic. Our results identify a valuable model and behavioural approach to advance mechanistic understandings of the trajectory of cognitive dysfunction following stroke, and provide a platform with which to identify novel therapies. Moreover, our work further strengthen the need for preclinical animal studies to assess different species and injury models that are critically needed for better modelling clinical stroke and truly enabling the translation of research targeting cognitive symptoms.

Maternal immune activation targeted to a window of parvalbumin interneuron development improves spatial working memory: Implications for autism

Maternal immune activation (MIA) increases risk for neuropsychiatric disorders such as autism spectrum disorder (ASD) in offspring later in life through unknown causal mechanisms. Growing evidence implicates parvalbumin-containing GABAergic interneurons as a key target in rodent MIA models. We targeted a specific neurodevelopmental window of parvalbumin interneurons in a mouse MIA model to examine effects on spatial working memory, a key domain in ASD that can manifest as either impairments or improvements both clinically and in animal models.Pregnant dams received three consecutive intraperitoneal injections of Polyinosinic:polycytidylic acid (poly(I:C), 5 mg/kg) at gestational days 13, 14 and 15. Spatial working memory was assessed in young adult offspring using touchscreen operant chambers and the Trial-Unique Non-matching to Location (TUNL) task. Anxiety, novelty seeking and short-term memory were assessed using Elevated Plus Maze (EPM) and Y-maze novelty preference tasks. Fluorescent immunohistochemistry was used to assess hippocampal parvalbumin cell density, intensity and co-expression with perineuronal nets. qPCR was used to assess the expression of putatively implicated gene pathways.MIA targeting a window of parvalbumin interneuron development increased spatial working memory performance on the TUNL touchscreen task which was not influenced by anxiety or novelty seeking behaviour. The model reduced fetal mRNA levels of Gad1 and adult hippocampal mRNA levels of Pvalb and the distribution of low intensity parvalbumin interneurons was altered.We speculate a specific timing window for parvalbumin interneuron development underpins the apparently paradoxical improved spatial working memory phenotype found both across several rodent models of autism and clinically in ASD.

Acute NMDA receptor antagonism impairs working memory performance but not attention in rats-Implications for the NMDAr hypofunction theory of schizophrenia.

Cognitive deficits in schizophrenia, which include impairments in working memory and attention, represent some of the most disabling symptoms of this complex psychiatric condition, and lack effective treatments. NMDA receptor (NMDAr) hypofunction is a strong candidate mechanism underlying schizophrenia pathophysiology, and has been modeled preclinically using acute administration of NMDAr antagonists to rodents to investigate biological mechanisms underpinning cognitive dysfunction. However, whether and how NMDAr hypofunction specifically influences all affected cognitive domains is unclear. Here we studied the effects of the NMDAr antagonist MK-801 (dizocilpine) on tasks of attention and working memory in rats using automated touchscreen chambers. Adult male Wistar rats were trained to perform the trial-unique nonmatching to location (TUNL) task of spatial working memory, or the 5-choice serial reaction time task (5CSRTT) of attention. Once trained, rats received injection of vehicle (saline) or low-dose MK-801 (0.06 mg/kg sc) 10 min prior to commencing test sessions. MK-801 significantly impaired working memory, as evidenced by reduced performance accuracy on the TUNL task (p < .0001), compared with vehicle. However, we found no significant effects on attentional processing or perseveration on the 5CSRTT. Additional measures indicated that MK-801 impaired behavioral flexibility in the TUNL task, and decreased response inhibition in both tasks. Using the automated touchscreen system to measure different cognitive functions under the same testing environment, we demonstrate that spatial working memory, response inhibition, and behavioral flexibility are more vulnerable to NMDAr hypofunction than attentional processing. This may have implications for the NMDAr hypofunction hypothesis of schizophrenia. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Task phase-specific involvement of the rat posterior parietal cortex in performance of the TUNL task.

The posterior parietal cortex (PPC) participates in cognitive processes including working memory (WM), sensory evidence accumulation, and perceptually guided decision making. However, surprisingly little work has used temporally precise manipulations to dissect its role in different epochs of behavior taking place over short timespans, such as WM tasks. As a result, a consistent view of the temporally precise role of the PPC in these processes has not been described. In the present study, we investigated the temporally specific role of the PPC in the Trial-Unique, Nonmatching-to-Location (TUNL) task, a touchscreen-based, visuospatial WM task that relies on the PPC. To disrupt PPC activity in a temporally precise manner, we applied mild intracranial electrical stimulation (ICES). We found that intra-PPC ICES (100 μA) significantly impaired accuracy in TUNL without significantly altering response latency. Moreover, we found that the impairment was specific to ICES applied during the delay and test phases of TUNL. Consistent with previous reports showing delay- and choice-specific neuronal activity in the PPC, the results provide evidence that the rat PPC is required for maintaining memory representations of stimuli over a delay period as well as for making successful comparisons and choices between test stimuli. In contrast, the PPC appears not to be critical for initial encoding of sample stimuli. This pattern of results may indicate that early encoding of visual stimuli is independent of the PPC or that the PPC becomes engaged only when visual stimuli are spatially complex or involve memory or decision making.

ChABC infusions into medial prefrontal cortex, but not posterior parietal cortex, improve the performance of rats tested on a novel, challenging delay in the touchscreen TUNL task.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that surround subsets of neurons throughout the central nervous system (CNS). They are made up of chondroitin sulfate proteoglycans (CSPGs), hyaluronan, tenascin-R, and many other link proteins that together make up their rigid and lattice-like structure. Modulation of PNNs can alter synaptic plasticity and thereby affect learning, memory, and cognition. In the present study, we degraded PNNs in the medial prefrontal (mPFC) and posterior parietal (PPC) cortices of Long–Evans rats using the enzyme chondroitinase ABC (ChABC), which cleaves apart CSPGs. We then measured the consequences of PNN degradation on spatial working memory (WM) with a trial-unique, non-matching-to location (TUNL) automated touchscreen task. All rats were trained with a standard 6 sec delay and 20 sec inter-trial interval (ITI) and then tested under four different conditions: a 6 sec delay, a variable 2 or 6 sec delay, a 2 sec delay with a 1 sec ITI (interference condition), and a 20 sec delay. Rats that received mPFC ChABC treatment initially performed TUNL with higher accuracy, more selection trials completed, and fewer correction trials completed compared to controls in the 20 sec delay condition but did not perform differently from controls in any other condition. Rats that received PPC ChABC treatment did not perform significantly differently from controls in any condition. Posthumous immunohistochemistry confirmed an increase in CSPG degradation products (C4S stain) in the mPFC and PPC following ChABC infusions while WFA staining intensity and parvalbumin positive neuron number were decreased following mPFC, but not PPC, ChABC infusions. These findings suggest that PNNs in the mPFC play a subtle role in spatial WM, but PNNs in the PPC do not. Furthermore, it appears that PNNs in the mPFC are involved in adapting to a challenging novel delay, but that they do not play an essential role in spatial WM function.

MK-801 impairs working memory on the Trial-Unique Nonmatch-to-Location test in mice, but this is not exclusively mediated by NMDA receptors on PV+ interneurons or forebrain pyramidal cells

NMDA receptors (NMDAr) are widely expressed throughout the brain on many cell types, and loss of function of these receptors (ie: NMDAr hypofunction) is a candidate mechanism explaining working memory impairment in schizophrenia. However, the cellular source driving the working memory deficits caused by NMDAr hypofunction has not been explored. The aim of this study was to assess the contribution of NMDAr on pyramidal cells and parvalbumin (PV+) interneurons to impairments in working memory induced by NMDAr hypofunction. We excised GluN1 - the gene encoding the obligatory subunit of the NMDAr - from PV + interneurons or CaMKIIα+ pyramidal cells using Cre-lox technology. Adult male PV GluN1 KO (n = 10) and CaMKIIα GluN1 KO mice (n = 9) and WT controls (n = 10 and n = 13) were trained to perform the Trial-Unique Nonmatching-to-Location (TUNL) task of working memory. Once trained, mice received the NMDAr antagonist MK-801 (0.1 and 0.3 mg/kg ip), and working memory assessed. Neither task acquisition nor working memory differed between the two transgenic lines and WT littermates. MK-801 dose-dependently decreased working memory accuracy in all strains (p < 0.001). PV GluN1 KO mice were sensitised to the impairing effects of MK-801 (p = 0.04), whereas CaMKIIα GluN1 KO mice showed equivalent working memory deficits as WT. Developmental NMDAr hypofunction at either PV+ interneurons or forebrain pyramidal cells is not sufficient to impair working memory, and neither of these cell types exclusively mediates working memory impairment caused by NMDAr antagonism. Reduced NMDAr signalling at PV+ interneurons could predispose circuits to NMDAr hypofunction magnifying deficits in working memory.

The mGluR2/3 agonist LY379268 reverses NMDA receptor antagonist effects on cortical gamma oscillations and phase coherence, but not working memory impairments, in mice.

Abnormalities in neural oscillations that occur in the gamma frequency range (30-80 Hz) may underlie cognitive deficits in schizophrenia. Both cognitive impairments and gamma oscillatory disturbances can be induced in healthy people and rodents by administration of N-methyl-D-aspartate receptor (NMDAr) antagonists. We studied relationships between cognitive impairment and gamma abnormalities following NMDAr antagonism, and attempted to reverse deficits with the metabotropic glutamate receptor type 2/3 (mGluR2/3) agonist LY379268. C57/Bl6 mice were trained to perform the Trial-Unique Nonmatching to Location (TUNL) touchscreen test for working memory. They were then implanted with local field potential (LFP) recording electrodes in prefrontal cortex and dorsal hippocampus. Mice were administered either LY379268 (3 mg/kg) or vehicle followed by the NMDAr antagonist MK-801 (0.3 or 1 mg/kg) or vehicle prior to testing on the TUNL task, or recording LFPs during the presentation of an auditory stimulus. MK-801 impaired working memory and increased perseveration, but these behaviours were not improved by LY379268 treatment. MK-81 increased the power of ongoing gamma and high gamma (130-180 Hz) oscillations in both brain regions and regional coherence between regions, and these signatures were augmented by LY379268. However, auditory-evoked gamma oscillation deficits caused by MK-801 were not affected by LY379268 pretreatment. NMDA receptor antagonism impairs working memory in mice, but this is not reversed by stimulation of mGluR2/3. Since elevations in ongoing gamma power and regional coherence caused by MK-801 were improved by LY379268, it appears unlikely that these specific oscillatory abnormalities underlie the working memory impairment caused by NMDAr antagonism.

Touchscreen testing reveals clinically relevant cognitive abnormalities in a mouse model of schizophrenia lacking metabotropic glutamate receptor 5

Metabotropic glutamate receptor 5 (mGlu5) has been implicated in certain forms of synaptic plasticity and cognitive function. mGlu5 knockout (KO) mice and mGlu5 antagonists have been previously used to study the pathophysiology of schizophrenia as they have been shown respectively to display or induce endophenotypes relevant to schizophrenia. While schizophrenia presents with generalized cognitive impairments, the cognitive phenotype of mice lacking mGlu5 has so far only been explored using largely hippocampal-dependent spatial and contextual memory tasks. To address this, we used a touchscreen system to assess mGlu5 KO mice for pairwise visual discrimination, reversal learning, and extinction of an instrumental response requiring no discrimination. Furthermore, we tested the role of mGlu5 in working memory using the Trial-Unique Non-Matching to Location (TUNL) task utilizing pharmacological ablation. mGlu5 KO mice were impaired on discrimination learning, taking longer to reach criterion and requiring more correction learning trials. Performance on reversal learning was also impaired, with mGlu5 KO mice demonstrating a perseverative phenotype. The mGlu5 KO mice responded at a higher rate during extinction, consistent with this perseverative profile. In contrast, wildtype mice treated acutely with an mGlu5 antagonist (MTEP) showed no deficits in a touchscreen task assessing working memory. The present study demonstrates learning and memory deficits as well as an increased perseverative phenotype following constitutive loss of mGlu5 in this mouse model of schizophrenia. These findings will inform translational approaches using this preclinical model and the pursuit of mGlu5 as therapeutic target for schizophrenia and other brain disorders.

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

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 1s or 6s delay between the sample and nonmatching stimuli. The estrous cycle of intact rats was monitored during the 4weeks 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 1s delay condition. Further, during the 6s 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.

An Automated, Experimenter-Free Method for the Standardised, Operant Cognitive Testing of Rats.

Animal models of human pathology are essential for biomedical research. However, a recurring issue in the use of animal models is the poor reproducibility of behavioural and physiological findings within and between laboratories. The most critical factor influencing this issue remains the experimenter themselves. One solution is the use of procedures devoid of human intervention. We present a novel approach to experimenter-free testing cognitive abilities in rats, by combining undisturbed group housing with automated, standardized and individual operant testing. This experimenter-free system consisted of an automated-operant system (Bussey-Saksida rat touch screen) connected to a home cage containing group living rats via an automated animal sorter (PhenoSys). The automated animal sorter, which is based on radio-frequency identification (RFID) technology, functioned as a mechanical replacement of the experimenter. Rats learnt to regularly and individually enter the operant chamber and remained there for the duration of the experimental session only. Self-motivated rats acquired the complex touch screen task of trial-unique non-matching to location (TUNL) in half the time reported for animals that were manually placed into the operant chamber. Rat performance was similar between the two groups within our laboratory, and comparable to previously published results obtained elsewhere. This reproducibility, both within and between laboratories, confirms the validity of this approach. In addition, automation reduced daily experimental time by 80%, eliminated animal handling, and reduced equipment cost. This automated, experimenter-free setup is a promising tool of great potential for testing a large variety of functions with full automation in future studies.

P.5.c.012 - Neuroprotective effect of pioglitazone in the 6-hydroxydopamine model of Parkinson’s disease

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.