Age-related decline of various cognitive functions in well-experienced male rats treated with the putative anti-aging compound (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine ((-)BPAP).

Aging-associated cognitive disorders lack proper medication. To meet this need translation-wise, modification of the animal models is also required. In the present study, effect of the putative anti-aging compound (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine ((-)BPAP, a deprenyl derivative) on age-related cognitive decline was investigated in experienced, aged Long-Evans rats. During their lifetime, animals had acquired knowledge in various cognitive assays. Their performance in these tests was then parallel followed from the age of 27 months until their death meanwhile half of them were treated with BPAP. Cognitive performance in various tasks showed different sensitivities/resistances to age-related impairment. Pot jumping performance (motor skill-learning) started to impair first, at 21 months of age, followed by decreasing performance in five-choice serial reaction time task (attention) at 26 months. Navigation performance in Morris water maze (spatial learning) started to decline at 31 months. Performance in a cooperation task (social cognition) started to decline the latest, at 34 months. Our findings suggest that in this process, the primary factor was the level of motivation to be engaged with the task and not losing the acquired knowledge. The average lifespan of the tested rat population was 36 months. BPAP could not improve the cognitive performance; neither could it prolong lifespan. A possible reason might be that dietary restriction and lifelong cognitive engagement had beneficial effects on cognitive capabilities and lifespan creating a "ceiling effect" for further improvement. The results confirmed that experienced animals provide a translationally relevant model to study age-related cognitive decline and measure the effect of putative anti-aging compounds.

Using Appetitive Motivation to Train Mice for Spatial Learning in the Barnes Maze.

The Barnes maze, a well-known spatial-learning paradigm, is based on the innate fear of rodents of large open spaces and their drive to hide. However, additional aversive stimuli (strong light and threatening sounds) are often necessary to provoke the hiding response while rendering the method cumbersome and more stressful. Our objective was to establish a Barnes maze-learning paradigm in mice using palatable food as a reward. After habituating male C57BL6/J or NMRI mice to the reward, the experimenter and the apparatus, either a slow (2 trials/day) or a massive conditioning schedule (4 trials/day), was run. Acquisition training was carried out until mice could locate the reward box with a maximum of one hole error. Then, the box was replaced to another location (reversal phase). Mice needed to relearn the new position with the same criterion. One week later, retention trials were performed. Both strains could reach the learning criteria; in the massive training within a shorter period. Spatial memory was demonstrated in the reversal and retention trials. Our results show that palatable food can be used as an efficient motivator to acquire allocentric navigation in the Barnes maze with the additional advantage of being less stressful.

Performance of the intracerebroventricularly injected streptozotocin Alzheimer's disease model in a translationally relevant, aged and experienced rat population.

The intracerebroventricularly (icv) injected streptozotocin (STZ) induced brain state is a widely used model of sporadic Alzheimer-disease (AD). However, data have been generated in young, naive albino rats. We postulate that the translationally most relevant animal population of an AD model should be that of aged rats with substantial learning history. The objective of the study was thus to probe the model in old rats with knowledge in various cognitive domains. Long-Evans rats of 23 and 10 months age with acquired knowledge in five-choice serial reaction time task (5-CSRTT), a cooperation task, Morris water-maze (MWM) and "pot-jumping" exercise were treated with 3 × 1.5 mg/kg icv. STZ and their performance were followed for 3 months in the above and additional behavioral assays. Both STZ-treated age groups showed significant impairment in the MWM (spatial learning) and novel object recognition test (recognition memory) but not in passive avoidance and fear conditioning paradigms (fear memory). In young STZ treated rats, significant differences were also found in the 5CSRTT (attention) and pot jumping test (procedural learning) while in old rats a significant increase in hippocampal phospho-tau/tau protein ratio was observed. No significant difference was found in the cooperation (social cognition) and pairwise discrimination (visual memory) assays and hippocampal ß-amyloid levels. STZ treated old animals showed impulsivity-like behavior in several tests. Our results partly coincide with partly deviate from those published on young, albino, unexperienced rats. Beside the age, strain and experience level of the animals differences can also be attributed to the increased dose of STZ, and the applied food restriction regime. The observed cognitive and non-cognitive activity pattern of icv. STZ in aged experienced rats call for more extensive studies with the STZ model to further strengthen and specify its translational validity.

Introduction of a pharmacological neurovascular uncoupling model in rats based on results of mice.

Our aim was to establish a pharmacologically induced neurovascular uncoupling (NVU) method in rats as a model of human cognitive decline. Pharmacologically induced NVU with subsequent neurological and cognitive defects was described in mice, but not in rats so far. We used 32 male Hannover Wistar rats. NVU was induced by intraperitoneal administration of a pharmacological "cocktail" consisting of N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH, a specific inhibitor of epoxyeicosatrienoic acid-producing epoxidases, 5 mg kg-1), L-NG-nitroarginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 10 mg kg-1) and indomethacin (a nonselective inhibitor of cyclooxygenases, 1 mg kg-1) and injected twice daily for 8 consecutive days. Cognitive performance was tested in the Morris water-maze and fear-conditioning assays. We also monitored blood pressure. In a terminal operation a laser Doppler probe was used to detect changes in blood-flow (CBF) in the barrel cortex while the contralateral whisker pad was stimulated. Brain and small intestine tissue samples were collected post mortem and examined for prostaglandin E2 (PGE2) level. Animals treated with the "cocktail" showed no impairment in their performance in any of the cognitive tasks. They had higher blood pressure and showed cca. 50% decrease in CBF. Intestinal bleeding and ulcers were found in some animals with significantly decreased levels of PGE2 in the brain and small intestine. Although we could evoke NVU by the applied mixture of pharmacons, it also induced adverse side effects such as hypertension and intestinal malformations while the treatment did not cause cognitive impairment. Thus, further refinements are still required for the development of an applicable model.

Corrigendum: Intracerebroventricularly Injected Streptozotocin Exerts Subtle Effects on the Cognitive Performance of Long-Evans Rats.

[This corrects the article DOI: 10.3389/fphar.2021.662173.].

Cognitive profiling and proteomic analysis of the modafinil analogue S-CE-123 in experienced aged rats.

The lack of novel cognitive enhancer drugs in the clinic highlights the prediction problems of animal assays. The objective of the current study was to test a putative cognitive enhancer in a rodent cognitive test system with improved translational validity and clinical predictivity. Cognitive profiling was complemented with post mortem proteomic analysis. Twenty-seven male Lister Hooded rats (26 months old) having learned several cognitive tasks were subchronically treated with S-CE-123 (CE-123) in a randomized blind experiment. Rats were sacrificed after the last behavioural procedure and plasma and brains were collected. A label-free quantification approach was used to characterize proteomic changes in the synaptosomal fraction of the prefrontal cortex. CE-123 markedly enhanced motivation which resulted in superior performance in a new-to-learn operant discrimination task and in a cooperation assay of social cognition, and mildly increased impulsivity. The compound did not affect attention, spatial and motor learning. Proteomic quantification revealed 182 protein groups significantly different between treatment groups containing several proteins associated with aging and neurodegeneration. Bioinformatic analysis showed the most relevant clusters delineating synaptic vesicle recycling, synapse organisation and antioxidant activity. The cognitive profile of CE-123 mapped by the test system resembles that of modafinil in the clinic showing the translational validity of the test system. The findings of modulated synaptic systems are paralleling behavioral results and are in line with previous evidence for the role of altered synaptosomal protein groups in mechanisms of cognitive function.

Intracerebroventricularly Injected Streptozotocin Exerts Subtle Effects on the Cognitive Performance of Long-Evans Rats.

Intracerebroventricularly injected streptozotocin (STZ)-induced learning impairment has been an increasingly used rat model of Alzheimer disease. The evoked pathological changes involve many symptoms of the human disease (cognitive decline, increase in ß-amyloid and phospho-tau level, amyloid plaque-like deposits). However, the model has predominantly been used with Wistar rats in the literature. The objective of the current study was to transfer it to Long-Evans rats with the ulterior aim to integrate it in a complex cognitive test battery where we use this strain because of its superior cognitive capabilities. We performed two experiments (EXP1, EXP2) with three months old male animals. At EXP1, rats were treated with 2 × 1.5 mg/kg STZ (based on the literature) or citrate buffer vehicle injected bilaterally into the lateral ventricles on days 1 and 3. At EXP2 animals were treated with 3 × 1.5 mg/kg STZ or citrate buffer vehicle injected in the same way as in EXP1 at days 1, 3, and 5. Learning and memory capabilities of the rats were then tested in the following paradigms: five choice serial reaction time test (daily training, started from week 2 or 8 post surgery in Exp1 or Exp2, respectively, and lasting until the end of the experiment); novel object recognition (NOR) test (at week 8 or 14), passive avoidance (at week 11 or 6) and Morris water-maze (at week 14 or 6). 15 or 14 weeks after the STZ treatment animals were sacrificed and brain phospho-tau/tau protein ratio and ß -amyloid level were determined by western blot technique. In EXP1 we could not find any significant difference between the treated and the control groups in any of the assays. In EXP2 we found significant impairment in the NOR test and elevated ß-amyloid level in the STZ treated group in addition to slower learning of the five-choice paradigm and a trend for increased phospho-tau/tau ratio. Altogether our findings suggest that the Long-Evans strain may be less sensitive to the STZ treatment than the Wistar rats and higher doses may be needed to trigger pathological changes in these animals. The results also highlight the importance of strain diversity in modelling human diseases.

Procognitive profiling of a serotonin 5-HT6 receptor antagonist in a complex model system in rats: A novel translational approach for clinical prediction.

INTRODUCTION: The serial clinical failures of novel cognitive enhancer candidates point out the lack of predictive power in the preceding animal experimentation. For a more predictive profiling of putative procognitive drugs in rodents, we recently elaborated a methodical approach which consists of three fundamental steps: 1. teaching various learning tasks representing different cognitive domains to the same cohort of animals with the aim to create a population with 'widespread knowledge'. 2. Applying a cognitive deficit-inducing intervention to transform this cohort of animals to a 'patient population'. 3. Testing putative procognitive drugs with a 'clinical trial-like' design on the wide spectrum of cognitive (dys)functions in the actual 'patient population'. The present study has been the first trial to test the feasibility and utility of the proposed system. METHODS: The population with 'widespread knowledge' consisted of 2 year old male Long-Evans rats with a learning history in five-choice serial reaction time task (5-CSRTT, attentional paradigm), Morris water maze (MWM, spatial learning), a cooperative task carried out in pairs (social learning), and a skill-learning task, "pot-jumping". For inducing cognitive deficit, thus creating a 'patient population' we increased the difficulty of the tasks. For the cognitive enhancer mechanism to test in the system we chose a serotonin 5-HT6 receptor antagonist compound, RO4368554. Animals were randomly assigned to vehicle- and drug treated groups based on their baseline learning performance and their response in a pilot test of increase in task difficulty. During the 13-day long treatment with 3 mg/kg ip. RO4368554 all the learning paradigms were repeatedly run with increased difficulty supplemented with a novel object recognition test (NOR, episodic memory). RESULTS: In the 5-CSRTT, reducing the stimulus duration from 1 s to 0.25 s caused a significant decrease in the percentage of correct responses (from 52 % to 31 % in the control group) which was not affected by the 5-HT6 receptor antagonist treatment (correct responses decreased from 58 % to 31 %). In the MWM, replacing the escape platform to a new location did not mean a hard challenge for the rats. Members of both groups could find it within a relatively short time: mean escape latencies were 83 s and 65 s at the first replacement trial and 58 s and 74 s at the second one in the control and drug-treated groups, respectively. In the cooperation paradigm, where the rats had to perform simultaneous nose-pokes to get a reward, task difficulty was increased by requiring two consecutive simultaneous nose-poking from the animals. This caused a fall in the percentage of successful trials in both groups (from 48 % to 12 % and from 50 % to 20 % in the saline - and drug-treated group, respectively), however, by the end of the treatment RO4368554-treated animals showed significantly higher performance (29 %) than saline treated rats (2%). The NOR test, carried out with a 5 -h delay, revealed poor recognition memory in both groups (discrimination index (DI) values were 0.13 and 0.06 for saline and RO4368554, respectively). Performance in the pot jumping test was also not improved by the drug-treatment. CONCLUSIONS: The applied study design allowed parallel measurements of the action of the test compound on several cognitive functions and to follow its time course. RO4368554 did not show notable effects on impaired attention and visual recognition; nor did it affect spatial and procedural learning, but it exerted beneficial effect on cooperative behaviour. The revealed activity pattern highlight the cognitive domain most sensitive to the particular drug effect and may give hints for further target validating and clinical studies.

Following of aging process in a new motor skill learning model, "pot jumping" in rats.

Impairment of procedural memory is a frequent and severe symptom in many neurological and psychiatric diseases as well as during aging. Our aim was to establish an assay in rats in which procedural learning and changes in performance can be studied on the long term. The work was done in the frame of a larger project aiming to establish a complex cognitive animal test battery of high translational value. The equipment was a 190-cm-diameter circular water tank where 12 flower pots were placed upside down in a circle with increasing distances (18-46 cm) between the adjacent ones. Male Lister Hooded and Long-Evans rats were allowed to move on the pots for 3 min. The arena was filled with shallow water to make the rats stay on the pots. Animals were obviously motivated to move around on the pots; however, the distance which required jumping (> 26 cm) meant a barrier for some of them. Development of motor skill was measured by the longest distance successfully spanned. A relatively flat bell-shaped age dependence was observed, with a peak at 13 months of age. A gradual decline in performance could be observed after the age of 20 months which preceded the appearance of overt physical weakness. Long-Evans rats showed more homogeneous performance and higher individual stability than Lister Hooded rats. The method is appropriate to study the development of motor learning and to follow its age-dependent changes. It may also serve as an assay for testing potential drugs for improving motor skills and/or procedural memory.

Translational Difficulties in Querying Rats on "Orientation".

The aim of this study was to translate the "orientation" query of the ADAS-Cog inventory to rats and to investigate whether they can determine which time of the day they are. For this purpose, we established a modified Morris water-maze navigation task where the escape platform was placed onto various locations at different times of the day: "morning", "noon" and "evening". In each of these sessions rats swam a "query" trial and a "confirmatory" trial, 30 min apart. Lister Hooded rats randomly chose among the three possible target locations, while Long Evans rats partly followed a win-stay strategy by preferring to visit first to the platform position of the previous session. Despite simplifying the task to a morning-evening discrimination, Lister Hooded rats continued searching by chance, while Long Evans rats switched to the mentally less demanding random strategy. We then inserted a board into the pool which required longer swimming path from the animals when they were correcting an initial wrong choice, but this modification did not result in a change in the above strategies. Lastly, in a separate group of Long-Evans rats, the training conditions were modified inasmuch an incorrect choice was definitely punished by impeding the animals to correct it and confining them to a platform-free part of the maze for the whole trial period. However, even these stricter conditions were not sufficient to make the rats distinguish times of the day. The observed lack of time discrimination may source from an evolutionary built in mechanism characteristic for the rat species or this ability may have only been lost in laboratory rats.

Selegiline reduces adiposity induced by high-fat, high-sucrose diet in male rats.

BACKGROUND AND PURPOSE: Incidence and severity of obesity are increasing worldwide, however, efficient and safe pharmacological treatments are not yet available. Certain MAO inhibitors reduce body weight, although their effects on metabolic parameters have not been investigated. Here, we have assessed effects of a widely used, selective MAO-B inhibitor, selegiline, on metabolic parameters in a rat model of diet-induced obesity. EXPERIMENTAL APPROACH: Male Long-Evans rats were given control (CON) or a high-fat (20%), high-sucrose (15%) diet (HFS) for 25 weeks. From week 16, animals were injected s.c. with 0.25 mg·kg-1 selegiline (CON + S and HFS + S) or vehicle (CON, HFS) once daily. Whole body, subcutaneous and visceral fat was measured by CT, and glucose and insulin tolerance were tested. Expression of glucose transporters and chemokines was assessed by quantitative RT-PCR. KEY RESULTS: Selegiline decreased whole body fat, subcutaneous- and visceral adiposity, measured by CT and epididymal fat weight in the HFS group, compared with HFS placebo animals, without influencing body weight. Oral glucose tolerance and insulin tolerance tests showed impaired glucose homeostasis in HFS and HFS + S groups, although insulin levels in plasma and pancreas were unchanged. HFS induced expression of Srebp-1c, Glut1 and Ccl3 in adipose tissue, which were alleviated by selegiline. CONCLUSIONS AND IMPLICATIONS: Selegiline reduced adiposity, changes in adipose tissue energy metabolism and adipose inflammation induced by HFS diet without affecting the increased body weight, impairment of glucose homeostasis, or behaviour. These results suggest that selegiline could mitigate harmful effects of visceral adiposity.