Leukotriene signaling as molecular correlate for cognitive heterogeneity in aging: an exploratory study.

Introduction: Aging is in general associated with a decline in cognitive functions. Looking more closely, there is a huge heterogeneity in the extent of cognitive (dys-)abilities in the aged population. It ranges from the population of resistant, resilient, cognitively unimpaired individuals to patients with severe forms of dementias. Besides the known genetic, environmental and life style factors that shape the cognitive (dys-)abilities in aging, the underlying molecular mechanisms and signals related to cognitive heterogeneity are completely unknown. One putative mechanism underlying cognitive heterogeneity might be neuroinflammation, exerted through microglia, the brain's innate immune cells, as neuroinflammation is central to brain aging and neurodegenerative diseases. Recently, leukotrienes (LTs), i.e., small lipid mediators of inflammation produced by microglia along aging and neurodegeneration, got in the focus of geroscience as they might determine cognitive dysfunctions in aging. Methods: Here, we analyzed the brain's expression of key components of the LT synthesis pathway, i.e., the expression of 5-lipoxygenase (5-Lox), the key enzyme in LT production, and 5-lipoxygenase-activating protein (FLAP) in young and aged rats. More specifically, we used a cohort of rats, which, although grown up and housed under identical conditions, developed into aged cognitively unimpaired and aged cognitively impaired traits. Results: Expression of 5-Lox was increased within the brain of aged rats with the highest levels detected in cognitively impaired animals. The number of microglia cells was higher in the aged compared to the young brains with, again, the highest numbers of 5-Lox expressing microglia in the aged cognitively impaired rats. Remarkably, lower cognitive scores in the aged rats associated with higher numbers of 5-Lox positive microglia in the animals. Similar data were obtained for FLAP, at least in the cortex. Our data indicate elevated levels of the LT system in the brain of cognitively impaired animals. Discussion: We conclude that 5-Lox expressing microglia potentially contribute to the age-related cognitive decline in the brain, while low levels of the LT system might indicate and foster higher cognitive functions and eventually cognitive reserve and resilience in aging.

Low-Affinity/High-Selectivity Dopamine Transport Inhibition Sufficient to Rescue Cognitive Functions in the Aging Rat.

The worldwide increase in cognitive decline, both in aging and with psychiatric disorders, warrants a search for pharmacological treatment. Although dopaminergic treatment approaches represent a major step forward, current dopamine transporter (DAT) inhibitors are not sufficiently specific as they also target other transporters and receptors, thus showing unwanted side effects. Herein, we describe an enantiomerically pure, highly specific DAT inhibitor, S-CE-123, synthetized in our laboratory. Following binding studies to DAT, NET and SERT, GPCR and kinome screening, pharmacokinetics and a basic neurotoxic screen, S-CE-123 was tested for its potential to enhance and/or rescue cognitive functions in young and in aged rats in the non-invasive reward-motivated paradigm of a hole-board test for spatial learning. In addition, an open field study with young rats was carried out. We demonstrated that S-CE-123 is a low-affinity but highly selective dopamine reuptake inhibitor with good bioavailability. S-CE-123 did not induce hyperlocomotion or anxiogenic or stereotypic behaviour in young rats. Our compound improved the performance of aged but not young rats in a reward-motivated task. The well-described impairment of the dopaminergic system in aging may underlie the age-specific effect. We propose S-CE-123 as a possible candidate for developing a tentative therapeutic strategy for age-related cognitive decline and cognitive dysfunction in psychiatric disorders.

Molecular species of oxidized phospholipids in brain differentiate between learning- and memory impaired and unimpaired aged rats.

Loss of cognitive function is a typical consequence of aging in humans and rodents. The extent of decline in spatial memory performance of rats, assessed by a hole-board test, reaches from unimpaired and comparable to young individuals to severely memory impaired. Recently, proteomics identified peroxiredoxin 6, an enzyme important for detoxification of oxidized phospholipids, as one of several synaptosomal proteins discriminating between aged impaired and aged unimpaired rats. In this study, we investigated several components of the epilipidome (modifications of phospholipids) of the prefrontal cortex of young, aged memory impaired (AI) and aged unimpaired (AU) rats. We observed an age-related increase in phospholipid hydroperoxides and products of phospholipid peroxidation, including reactive aldehydophospholipids. This increase went in hand with cortical lipofuscin autofluorescence. The memory impairment, however, was paralleled by additional specific changes in the aged rat brain epilipidome. There was a profound increase in phosphocholine hydroxides, and a significant decrease in phosphocholine-esterified azelaic acid. As phospholipid-esterified fatty acid hydroxides, and especially those deriving from arachidonic acid are both markers and effectors of inflammation, the findings suggest that in addition to age-related reactive oxygen species (ROS) accumulation, age-related impairment of spatial memory performance has an additional and distinct (neuro-) inflammatory component.

The Lack of Dopamine Transporter Is Associated With Conditional Associative Learning Impairments and Striatal Proteomic Changes.

Dopamine (DA) is critically involved in different functions of the central nervous system (CNS) including control of voluntary movement, affect, reward, sleep, and cognition. One of the key components of DA neurotransmission is DA reuptake by the DA transporter (DAT), ensuring rapid clearance of DA from the synaptic cleft. Thus, lack of DAT leads to persistent high extracellular DA levels. While there is strong evidence for a role of striatal dopaminergic activity in learning and memory processes, little is known about the contribution of DAT deficiency to conditional learning impairments and underlying molecular processes. DAT-knockout (DAT-KO) rats were tested in a set of behavioral experiments evaluating conditional associative learning, which requires unaltered striatal function. In parallel, a large-scale proteomic analysis of the striatum was performed to identify molecular factors probably underlying behavioral patterns. DAT-KO rats were incapable to acquire a new operant skill in Pavlovian/instrumental autoshaping, although the conditional stimulus-unconditional stimulus (CS-US) association seems to be unaffected. These findings suggest that DAT directly or indirectly contributes to the reduction of transference of incentive salience from the reward to the CS. We propose that specific impairment of conditional learning might be caused by molecular adaptations to the hyperdopaminergic state, presumably by dopamine receptor 1 (DRD1) hypofunction, as proposed by proteomic analysis. Whether DRD1 downregulation can cause cognitive deficits in the hyperdopaminergic state is the subject of discussion, and further studies are needed to answer this question. This study may be useful for the interpretation of previous and the design of future studies in the dopamine field.

Reinstatement of synaptic plasticity in the aging brain through specific dopamine transporter inhibition.

Aging-related neurological deficits negatively impact mental health, productivity, and social interactions leading to a pronounced socioeconomic burden. Since declining brain dopamine signaling during aging is associated with the onset of neurological impairments, we produced a selective dopamine transporter (DAT) inhibitor to restore endogenous dopamine levels and improve cognitive function. We describe the synthesis and pharmacological profile of (S,S)-CE-158, a highly specific DAT inhibitor, which increases dopamine levels in brain regions associated with cognition. We find both a potentiation of neurotransmission and coincident restoration of dendritic spines in the dorsal hippocampus, indicative of reinstatement of dopamine-induced synaptic plasticity in aging rodents. Treatment with (S,S)-CE-158 significantly improved behavioral flexibility in scopolamine-compromised animals and increased the number of spontaneously active prefrontal cortical neurons, both in young and aging rodents. In addition, (S,S)-CE-158 restored learning and memory recall in aging rats comparable to their young performance in a hippocampus-dependent hole board test. In sum, we present a well-tolerated, highly selective DAT inhibitor that normalizes the age-related decline in cognitive function at a synaptic level through increased dopamine signaling.

CE-123, a novel dopamine transporter inhibitor, attenuates locomotor hyperactivity and improves cognitive functions in rat model of fetal alcohol spectrum disorders.

Perinatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), usually first diagnosed in childhood, that are characterized by hyperactivity, impulsivity and learning and memory disability, among others. To test the hypothesis that dopamine signaling is one of the main factors underlying these impairments, a new atypical dopamine transporter (DAT) inhibitor, CE-123 (1, 3 or 10 mg/kg) was assessed for its potential to overcome the ethanol-induced behavioral effects in a rat model of FASD. In the present study, neonatal rats were exposed to alcohol intubations across the neonatal period (postnatal day (PND)4-9, the third trimester equivalent of human gestation) and, after weaning, the animals (male rats) were assigned randomly to three groups. The first group was tested at PND21 (hyperactivity test). A second group was tested at PND45 (anxiety test), at PND47 (locomotor activity test), at PND49 (spatial cognitive test in the Barnes maze) and PND50 (reversal learning in the Barnes maze). The third group was tested at PND50 (dopamine receptor mRNA expression). Our results support the hypothesis that dopamine signaling is associated with FASD because the dopamine (D1, D2 and D5) receptor mRNA expression was altered in the striatum, hippocampus and prefrontal cortex in adult rats exposed to ethanol during neonatal period. CE-123 (3 and 10 mg/kg) inhibited the hyperactivity and ameliorated (10 mg/kg) the impairment of reversal learning in alcohol-exposed rats. Thus, these findings provide support that CE-123 may be a useful intervention for same of the deficits associated with neonatal ethanol exposure.

Differences in Hypothalamic Lipid Profiles of Young and Aged Male Rats With Impaired and Unimpaired Spatial Cognitive Abilities and Memory.

Lipids play a major role for several brain functions, including cognition and memory. There is a series of work on individual lipids showing involvement in memory mechanisms, a concise lipidome was not reported so far. Moreover, there is no evidence for age-related memory decline and there is only work on brain of young vs. aging animals. Aging animals, however, are not a homogeneous group with respect to memory impairments, thus animals with impaired and unimpaired memory can be discriminated. Following recent studies of hippocampal lipid profiles and hypothalamus controlled hormone profiles, the aim of this study was to compare hypothalamic, lipidomic changes in male Sprague-Dawley rats between young (YM), old impaired (OMI) and old unimpaired (OMU) males. Grouping criterions for aged rats were evaluated by testing them in a spatial memory task, the hole-board. YMs were also tested. Subsequently brains were removed, dissected and hypothalami were kept at -80°C until sample preparation and analysis on liquid chromatography / mass spectrometry (LC-MS). Significant differences in the amounts of a series of lipids from several classes could be detected between young and aged and between OMI and OMU. A large number of lipids were increased in OMI and a smaller number in OMU as compared to young rats. Differences of lipid ratios (log2 of ratio) between OMI and OMU consisted of glycerophosphocholines (aPC 36:2 and 36:3; PC 34:0, 36:1, 36:3 and 40:2); Glycerophosphoethanolamines (aPE 34:2, 38:5 and 40:5; LPE 18:1, 20:1, 20:4, 22:4 and 22:6; PE36:1 and 38:4); glycerophosphoserines (PS 36:1, 40:4, and 40:6); triacylglycerol TG 52:4; ceramide Cer 17:2 and sphingomyelin SM 20:0. Thus, hypothalamic lipid profiles across different lipid classes discriminate aged male animals into OMU and OMI. The underlying mechanisms may be related to different functional networks of lipids in memory mechanisms and differences in metabolic processes. The study underlines the importance of lipidomics in the pathophysiology of age-related cognitive decline. The necessity of evaluating the cognitive status of aged subjects by behavioral tests results in more specific detection of critical lipids in memory decline, on which now can be focused in subsequent memory studies in animals and humans.

Doublecortin and IGF-1R protein levels are reduced in spite of unchanged DNA methylation in the hippocampus of aged rats.

The aim of the current study was to investigate whether doublecortin (DCX), insulin-like growth factor receptor 1 (IGF-1R) and metabotropic glutamate receptor 5 (mGluR5) levels are indeed modified in the aging rat hippocampal individual subareas (rather than total hippocampal tissue as in previous reports) at the protein and mRNA level and whether the methylation status contributes to these changes. Since the aging population is not homogeneous in terms of spatial memory performance, we examined whether changes in DCX, IGF-1R and mGluR5 are linked to cognitive aging. Aged (22 months) male Sprague Dawley rats were trained in the hole-board, a spatial memory task, and were subdivided according to performance to aged impaired and aged unimpaired groups. Age- and memory performance-dependent changes in mRNA steady-state levels, protein levels and DNA methylation status of DCX, IGF-1R and mGluR5 were evaluated by RT-PCR, immunoblotting and bisulfite pyrosequencing. Extending previous findings, we detected decreased DCX protein and mRNA levels in dentate gyrus (DG) of aged animals. IGF-1 signaling is a key event and herein we show that mRNA levels for IGF-1R were unchanged although reduced at the protein level. This finding may simply reflect that these protein levels are regulated at the level of protein synthesis as well as protein degradation. We provide evidence that promoter methylation is not involved in regulation of mRNA and protein levels of DCX, IGF-1R and mGluR5 during aging. Moreover, there was no significant difference between aged rats with impaired and aged rats with unimpaired memory at the protein and mRNA level. Findings propose that changes in the abovementioned protein levels may not be relevant for performance in the spatial memory task used in aged rats.

Striatal Transcriptome Reveals Differences Between Cognitively Impaired and Unimpaired Aged Male Rats.

Cognitive processes require striatal activity. The underlying molecular mechanisms are widely unknown. For this reason the striatal transcriptome of young (YM), aged cognitively impaired (OMB), and unimpaired (OMG) male rats was analyzed. The global comparison of transcripts reveal a higher number of differences between OMG and YM as compared to OMB and YM. Hierarchical clustering detects differences in up- and down-regulated gene clusters in OMG and OMB when compared to YM. In OMG we found more single genes to be specifically regulated in this group than in OMB when compared to young. These genes were considered as cognition specific, whereas genes shared in OMG and OMB were considered as age specific. OMB specific up-regulated genes are related to negative control of cell differentiation and transcription (Hopx), to phagocytosis (Cd202) and cell adhesion (Pcdhb21), whereas down-regulated genes are related to associative learning, behavioral fear response and synaptic transmission (Gabra5). OMG specific up-regulated genes are in the context of maintenance of transcription and estrogen receptor signaling (Padi2, Anxa3), signal transduction [Rassf4, Dock8)], sterol regulation (Srebf1), and complement activity (C4a, C4b). Down-regulated genes are related to lipid oxidation reduction processes (Far2) and positive regulation of axon extension (Islr2). These relations were supported by pathway analysis, which reveals cholesterol metabolism processes in both aged group and cholesterol biosynthesis specifically in OMG; adipogenesis and focal adhesion in OMB. In OMG glucuronidation, estrogen metabolism, inflammatory responses and TGF beta signaling where detected as specific for this group. Signal transduction of the sphingosine-1-phospate-receptor (S1P) receptor was the main pathway difference in the comparison of OMB and OMG with downregulated genes in the first group. This difference could also be observed in the OMB vs. YM comparison but not in the OMG vs. YM analysis. Thus, an up-regulation of cognition related genes could be observed in OMG compared to OMB rats. The S1P pathway discriminated between OMB and OMG as well as between OMB and OMG. Since this pathway has been described as essential for cognitive processes in the striatum of mice, it may, among steroid hormone signaling, significantly contribute to the maintenance of cognitive processes in OMG.

Dentate Gyrus Peroxiredoxin 6 Levels Discriminate Aged Unimpaired From Impaired Rats in a Spatial Memory Task.

Similar to humans, the normal aged rat population is not homogeneous in terms of cognitive function. Two distinct subpopulations of aged Sprague-Dawley rats can be identified on the basis of spatial memory performance in the hole-board paradigm. It was the aim of the study to reveal protein changes relevant to aging and spatial memory performance. Aged impaired (AI) and unimpaired (AU) male rats, 22-24 months old were selected from a large cohort of 160 animals; young animals served as control. Enriched synaptosomal fractions from dentate gyrus from behaviorally characterized old animals were used for isobaric tags labeling based quantitative proteomic analysis. As differences in peroxiredoxin 6 (PRDX6) levels were a pronounced finding, PRDX6 levels were also quantified by immunoblotting. AI showed impaired spatial memory abilities while AU performed comparably to young animals. Our study demonstrates substantial quantitative alteration of proteins involved in energy metabolism, inflammation and synaptic plasticity during aging. Moreover, we identified protein changes specifically coupled to memory performance of aged rats. PRDX6 levels clearly differentiated AI from AU and levels in AU were comparable to those of young animals. In addition, it was observed that stochasticity in protein levels increased with age and discriminate between AI and AU groups. Moreover, there was a significantly higher variability of protein levels in AI. PRDX6 is a member of the PRDX family and well-defined as a cystein-1 PRDX that reduces and detoxifies hydroxyperoxides. It is well-known and documented that the aging brain shows increased active oxygen species but so far no study proposed a potential target with antioxidant activity that would discriminate between impaired and unimpaired memory performers. Current data, representing so far the largest proteomics data set in aging dentate gyrus (DG), provide the first evidence for a probable role of PRDX6 in memory performance.

Age and cognitive status dependent differences in blood steroid and thyroid hormone concentrations in intact male rats.

BACKGROUND: Age-dependent alterations of hormonal states have been considered to be involved in age related decline of cognitive abilities. Most of the studies in animal models are based on hormonal substitution in adrenal- and/or gonadectomized rodents or infusion of steroid hormones in intact rats. Moreover, the manipulations have been done timely, closely related to test procedures, thus reflecting short-term hormonal mechanisms in the regulation of learning and memory. Here we studied whether more general states of steroid and thyroid hormone profiles, independent from acute experiences, may possibly reflect long-term learning capacity. A large cohort of aged (17-18 months) intact male rats were tested in a spatial hole-board learning task and a subset of inferior and superior learners was included into the analysis. Young male adult rats (16 weeks of age) were also tested. Four to 8 weeks after testing blood plasma samples were taken and hormone concentrations of a variety of steroid hormones were measured by gas chromatography-tandem mass spectrometry or radioimmunoassay (17ß-estradiol, thyroid hormones). RESULTS: Aged good learners were similar to young rats in the behavioral task. Aged poor learners but not good learners showed higher levels of triiodothyronine (T3) as compared to young rats. Aged good learners had higher levels of thyroid stimulating hormone (TSH) than aged poor learning and young rats. Both aged good and poor learners showed significantly reduced levels of testosterone (T), 4-androstenedione (4A), androstanediol-3α,17ß (AD), dihydrotestosterone (DHT), 17-hydroxyprogesterone (17OHP), higher levels of progesterone (Prog) and similar levels of 17ß-estradiol (E2) as compared to young rats. The learning, but not the memory indices of all rats were significantly and positively correlated with levels of dihydrotestosterone, androstanediol-3α,17ß and thyroxine (T4), when the impacts of age and cognitive division were eliminated by partial correlation analyses. CONCLUSION: The correlation of hormone concentrations of individuals with individual behavior revealed a possible specific role of these androgen and thyroid hormones in a state of general preparedness to learn.

Moderate differences in common feeding diets change lipid composition in the hippocampal dentate gyrus and affect spatial cognitive flexibility in male rats.

There is growing evidence that lipids play a fundamental role in neuronal plasticity and learning and memory. Effects of nutrition on brain lipid composition and neuronal functioning are known, but the feeding interventions are often severe and may not reflect nutritional effects below clinical relevance. Therefore, we tested two commercially available rat feeding diets with only moderate differences in the food compositions, a standard diet (gross energy metabolizable 12.8 MJ/kg) and a energy reduced diet (gross energy metabolizable 8.9 MJ/kg) on possible effects upon dentate gyrus lipid composition, spatial learning and memory in a water maze and corticosterone release (blood serum concentrations) in adult male rats. Rats were fed with the standard diet up to an age of 8 weeks. One group was further fed with the standard and another with the energy reduced diet until an age of 5 months. We did not found differences in serum corticosterone levels. We found group differences in a variety of lipids in the hippocampal dentate gyrus.. Most of the lipid levels were lower in energy reduced diets, namely glycerophosphoethanolamines, sphingomyelins and hexosyceramides, whereas some ceramides (Cer18:0 and Cer24:1) and glycerophosphocholines (PC34:3 and PC36:2) were upregulated compared to the standard diet group. The performance in a common reference memory water maze task was not different between groups, however during reversal learning (platform in a different position) after the initial training, the standard diet fed rats learned better and spatial memory was improved compared to the energy reduced diet group. Thus, moderate differences in feeding diets have effects specifically upon spatial cognitive flexibility. Possible relations between differences in lipid composition and cognitive flexibility are discussed.

Dopamine type 1- and 2-like signaling in the modulation of spatial reference learning and memory.

Spatial reference memory is known to be modulated by the dopaminergic system involving different brain regions. Here, we sought to identify the contribution of D1 (D1R) and D2 (D2R)-like dopamine receptor signaling on learning and memory in a food rewarded hole-board task by intracerebroventricular infusing D1R- and D2R- like receptor agonists (SKF-81297 and Sumanirole) and antagonists (SCH 23390 and Remoxipride) once 30 min prior to daily training sessions. D1R agonism induced persistent enhancement of performance, whereas D1R antagonism impaired reference memory formation. D2R agonist and antagonist exerted no effects. Phase specific comparisons revealed an enhancement of spatial acquisition in the presence of the D1R but not D2R agonism on acquisition, but not during retention. Since task difficulty might skew dopamine-induced improvements in learning and memory, we tested the D1R agonist in the hole-board task with increased difficulty. Drug treated animals performed significantly better during all training phases, with results better resolved than in the easy task. Additionally, proteomic analysis of the prefrontal cortex revealed ninety six proteins to be regulated by D1R agonism, from which 35 were correlated with behavioral performance. Obtained targets were grouped by function, showing synaptic transmission, synaptic remodeling, and dendritic spine morphology as the major functional classes affected. In sum, we find that activation of D1R signaling during spatial acquisition and retention improved reference memory index, depended on the task difficulty, and altered the proteome landscape of the prefrontal cortex indicative of massive organizational synaptic restructuring.

Moderate Differences in Feeding Diets Largely Affect Motivation and Spatial Cognition in Adult and Aged but Less in Young Male Rats.

Nutrition can have significant effects on behavior and cognitive processes. Most of the studies related to this use extremely modified diets, such as high fat contents or the exclusion of distinct components needed for normal development and bodily homeostasis. Here we report significant effects of diets with moderate differences in compositions on food rewarded spatial learning in young (3-4 months), adult (6-7 months), and aged (17-18 months) rats. Young rats fed with a lower energy diet showed better performance only during aquisition of the spatial task when compared to rats fed with a standard diet. Adult rats (6-7 months) fed with a standard diet performed less well in the spatial learning task, than rats fed with lower energy diet. Aged rats fed with a lower energy diet (from 13 to 18 months of age) performed better during all training phases, as in a previous test when they were adult and fed with a standard diet. This difference could only be partly explained by lower motivation to search for food in the first test. Correspondingly, the variability of individual performance was significantly higher and increased over trials in adult rats fed with the standard diet as compared to adult rats fed with lower energy diet. Thus, moderate changes in feeding diets have large effects on motivation and cognition in elderly and less in young rats in a food rewarded spatial learning task. Therefore, nutrition effects upon food rewarded spatial learning and memory should be considered especially in aging studies.

A daily single dose of a novel modafinil analogue CE-123 improves memory acquisition and memory retrieval.

Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to characterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked uptake of [3H] dopamine (IC50 = 4.606 µM) while effects on serotonin (SERT) and the norepinephrine transporter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval, represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1, CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory enhancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic for impairments of cognitive functions.

Evaluating Working Memory on a T-maze in Male Rats.

Working memory is short-term memory, so temporal improvement does not reflect the consolidation of a memory trace, rather the functionality of the underlying neuronal circuits and molecular signaling cascades. The administration of drugs-either one-time or through daily injection-can elucidate the underlying mechanisms. The T-maze is especially suitable for studying dopamine-dependent working memory, since it is less stressful than other tests, for example, water maze-based paradigms ( Bezu et al., 2016 and 2017). Here, we present a training protocol for evaluating the underlying mechanisms that lead to the development of spatial working memory in rats. Our approach uses a T-maze, and it can be used to get high temporal resolution.

Behavioral tagging: Synaptic event or cellular alteration?

Recently a number of studies were published addressing the translation of the synaptic tagging and capture hypothesis (STCH), developed and tested in hippocampal slices, into learning and long-term memory mechanisms in intact animals. Based on behavioral observations, these studies show similar results as in the original STCH experiments. A weak memory, induced by weak training in one hippocampus dependent behavioral task is converted into a long-term memory, by timely related induction of a long-term memory, induced by strong training in a second hippocampus dependent task. The induction of long-term memory should result in the synthesis of plasticity related proteins (PRP), which can be captured by the synapses involved in the weak memory trace and thus be converted into a long-term memory. Here it is outlined, that these results can alternatively be explained by non-synaptic mechanisms like changes in neuronal intrinsic excitability (IE). The plasticity of short and long-term IE show some common features with synaptic plasticity. Thus, behavioral tagging experiments, purely based on behavioral observations, do not necessarily support that synaptic tagging and capture mechanisms are involved in memory formations, unless it has been shown that the weak and strong training protocols result in synaptic early and late potentiation, or the STCH could be extended to short and long-term forms of intrinsic excitability.

Reduced Levels of the Synaptic Functional Regulator FMRP in Dentate Gyrus of the Aging Sprague-Dawley Rat.

Fragile X mental retardation protein (FMRP) encoded by Fragile X mental retardation 1 (FMR1) gene is a RNA-binding regulator of mRNA translation, transport and stability with multiple targets responsible for proper synaptic function. Epigenetic silencing of FMR1 gene expression leads to the development of Fragile X syndrome (FXS) that is characterized by intellectual disability and other behavioral problems including autism. In the rat FXS model, the lack of FMRP caused a deficit in hippocampal-dependent memory. However, the hippocampal changes of FMRP in aging rats are not fully elucidated. The current study addresses the changes in FMRP levels in dentate gyrus (DG) from young (17 weeks) and aging (22 months) Sprague - Dawley rats. The aging animal group showed significant decline in spatial reference memory. Protein samples from five rats per each group were analyzed by quantitative proteomic analysis resulting in 153 significantly changed proteins. FMRP showed significant reduction in aging animals which was confirmed by immunoblotting and immunofluorescence microscopy. Furthermore, bioinformatic analysis of the differential protein dataset revealed several functionally related protein groups with individual interactions with FMRP. These include high representation of the RNA translation and processing machinery connected to FMRP and other RNA-binding regulators including CAPRIN1, the members of Pumilio (PUM) and CUG-BP, Elav-like (CELF) family, and YTH N(6)-methyladenosine RNA-binding proteins (YTHDF). The results of the current study point to the important role of FMRP and regulation of RNA processing in the rat DG and memory decline during the aging process.

A Novel Dopamine Transporter Inhibitor CE-123 Improves Cognitive Flexibility and Maintains Impulsivity in Healthy Male Rats.

Reduced cognitive abilities are often characterized by an impairment of flexibility, i.e., the ability to switch from learned rules or categories that were important in certain contexts to different new modalities that rule the task. Drugs targeting the dopamine transporter (DAT) are widely used for their potential to enhance cognitive abilities. However, commercially available drugs are of limited specificity for DAT, blocking also noradrenaline and serotonine transporters, that can lead to unwanted side effects in healthy subjects. Therefore, we tested a newly synthetized compound (CE-123) with higher specificity for DAT in male rats in an attentional set-shifting task (ASST), that proves for cognitive flexibility and a 5-choice serial-reaction time task (5-CSRTT) assessing visuospatial attention and impulsivity. Treated rats at a dose of 0.3 and 1.0 but not 0.1 mg/kg bodyweight showed reduced extra-dimensional shifts in the ASST compared to controls indicating increased cognitive flexibility. Rats treated with R-Modafinil, a commercially available DAT inhibitor at a dose of 10 mg/kg bodyweight showed increased premature responses, an indicator of increased impulsivity, during a 10 s but not a 2.5, 5, or 7.5 s intertrial interval when compared to vehicle-treated rats in the 5-CSRTT. This was not found in rats treated with CE-123 at the same dose as for R-Modafinil. Visuospatial attention, except premature responses, did not differ between R-Modafinil and CE-123-treated rats and their respective controls. Thus, CE-123 increased cognitive flexibility with diminished impulsivity.

Spatial Working Memory in Male Rats: Pre-Experience and Task Dependent Roles of Dopamine D1- and D2-Like Receptors.

The dopaminergic system is known to be involved in working memory processed by several brain regions like prefrontal cortex (PFC), hippocampus, striatum. In an earlier study we could show that Levodopa but not Modafinil enhanced working memory in a T-maze only during the early phase of training (day 3), whereas the later phase remained unaffected. Rats treated with a higher dose performed better than low dose treated rats. Here we could more specifically segregate the contributions of dopamine type 1- and 2- like receptors (D1R; D2R) to the training state dependent modulation of spatial working memory by intracerebroventricular (ICV) application of a D1R-like (SKF81297) and D2R-like agonist (Sumanirole) and antagonist (SCH23390, Remoxipride) at a low and high dose through 3 days of training. The D1R-like-agonist at both doses enhanced working memory at day 1 but only in the low dose treated rats enhancement persists over training compared to control rats. Rats treated with a high dose of a D1R-like-antagonist show persistent enhancement of working memory over training, whereas in low dose treated rats no statistical difference at any time point could be determined compared to controls. The D2R-like-agonist at both doses does not show an effect at any time point when compared to control animals, whereas the D2R-like antagonist at a low dose enhanced working memory at day 2. For the most effective D1R-like agonist, we repeated the experiments in a water maze working memory task, to test for task dependent differences in working memory modulations. Treated rats at both doses did not differ as compared to controls, but the temporal behavioral performance of all groups was different compared to T-maze trained rats. The results are in line with the view that spatial working memory is optimized within a limited range of dopaminergic transmission, however suggest that these ranges vary during spatial training.