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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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Spatial reference memory is known to be modulated by the dopaminergic system involving different brain regions. Here, we sought to identify the contribution of D 1 (D1R) and D 2 (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., (Copyright © 2019. Published by Elsevier B.V.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A series of compounds have been shown to enhance cognitive function via the dopaminergic system and indeed the search for more active and less toxic compounds is continuing. It was therefore the aim of the study to synthetise and test a novel heterocyclic compound for cognitive enhancement in a paradigm for working memory. Specific and effective dopamine re-uptake inhibition DAT (IC50=4,1±0,8μM) made us test this compound in a radial arm maze (RAM) in the rat. CE-125 (4-((benzhydrylsulfinyl)methyl)-2-cyclopropylthiazole), was tested for dopamine (DAT), serotonin and norepinephrine re-uptake inhibition by a well-established system. The working memory index (WMI) was evaluated in male Sprague Dawley rats that were intraperitoneally injected with CE-125 (1 or 10mg/kg body weight). In order to evaluate basic neurotoxicity, the open field, elevated plus maze, rota rod studies and the forced swim test were carried out. Frontal cortex was taken at the last day of the RAM test and dopamine receptors D1R and D2R, DAT and phosphorylated DAT protein levels were determined. On the 10th day both doses were increasing the WMI as compared to the vehicle-treated group. In both, trained and treated groups, D1R levels were significantly reduced while D2R levels were unchanged. DAT levels were comparable between all groups while phosphorylated DAT levels were increased in the trained group treated with 1mg/kg body weight. CE-125 as a probably non-neurotoxic compound and specific reuptake inhibitor was shown to increase performance (WMI) and modulation of the dopaminergic system is proposed as a possible mechanism of action., (Copyright © 2017. Published by Elsevier B.V.)

A series of compounds targeting the dopamine transporter (DAT) haS been shown to improve memory performance most probably by re-uptake inhibition. Although specific DAT inhibitors are available, there is limited information about specificity, mechanism and in particular the effect on dopamine receptors. It was therefore the aim of the study to test the DAT inhibitor 4-(diphenyl-methanesulfinylmethyl)-2-methyl-thiazole (code: CE-111), synthetized in our laboratory for the specificity to target DAT, for the effects upon spatial memory and for induced dopamine receptor modulation. Re-uptake inhibition was tested for DAT (IC50=3.2μM), serotonin transporter, SERT (IC50=272291μM) and noradrenaline transporter, NET (IC50=174μM). Spatial memory was studied in the radial arm maze (RAM) in male Sprague-Dawley rats that were intraperitoneally injected with CE-111 (1 or 10mg/kg body weight). Performance in the RAM was improved using 1 and 10mg/kg body weight of CE-111. Training and treatment effects on presynaptic, postsynaptic and extrasynaptic D1 and D2- receptors and dopamine receptor containing complexes as well as on activated DAT were observed. CE-111 was crossing the blood-brain barrier comparable to modafinil and was identified as effective to improve memory performance in the RAM. Dopamine re-uptake inhibition along with modulations in dopamine receptors are proposed as potential underlying mechanisms., (Copyright © 2016. Published by Elsevier B.V.)

Cognition enhancing drugs often target the dopaminergic system, which is involved in learning and memory, including working memory that in turn involves mainly the prefrontal cortex and the hippocampus. In most animal models for modulations of working memory animals are pre-trained to a certain criterion and treated then acutely to test drugs effects on working memory. Thus, little is known regarding subchronic or chronic application of cognition enhancing drugs and working memory performance. Therefore we trained male rats over six days in a rewarded alternation test in a T-maze. Rats received daily injections of either modafinil or Levodopa (L-Dopa) at a lower and a higher dose 30min before training. Levodopa but not modafinil increased working memory performance during early training significantly at day 3 when compared to vehicle controls. Both drugs induced dose dependent differences in working memory with significantly better performance at low doses compared to high doses for modafinil, in contrast to L-Dopa where high dose treated rats performed better than low dose rats. Strikingly, these effects appeared only at day 3 for both drugs, followed by a decline in behavioral performance. Thus, a critical drug independent time window for dopaminergic effects upon working memory could be revealed. Evaluating the underlying mechanisms contributes to the understanding of temporal effects of dopamine on working memory performance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms., (Copyright © 2016. Published by Elsevier B.V.)

Several neurotransmitter receptors have been proposed to be involved in memory formation. However, information on receptor complexes (RCs) in the radial arm maze (RAM) is missing. It was therefore the aim of this study to determine major neurotransmitter RCs levels that are modulated by RAM training because receptors are known to work in homo-or heteromeric assemblies. Immediate early gene Arc expression was determined by immunohistochemistry to show if prefrontal cortices (PFC) and hippocampi were activated following RAM training as these regions are known to be mainly implicated in spatial memory. Twelve rats per group, trained and untrained in the twelve arm RAM were used, frontal cortices and hippocampi were taken, RCs in membrane protein were quantified by blue-native PAGE immunoblotting. RCs components were characterised by co-immunoprecipitation followed by mass spectrometrical analysis and by the use of the proximity ligation assay. Arc expression was significantly higher in PFC of trained as compared to untrained rats whereas it was comparable in hippocampi. Frontal cortical levels of RCs containing AMPA receptors GluA1, GluA2, NMDA receptors GluN1 and GluN2A, dopamine receptor D1, acetylcholine nicotinic receptor alpha 7 (nAChR-α7) and hippocampal levels of RCs containing D1, GluN1, GluN2B and nAChR-α7 were increased in the trained group; phosphorylated dopamine transporter levels were decreased in the trained group. D1 and GluN1 receptors were shown to be in the same complex. Taken together, distinct RCs were paralleling performance in the RAM which is relevant for interpretation of previous and design of future work on RCs in memory studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The current study was designed to examine learning-induced transformation of early-LTP into late-LTP. Recording electrodes were implanted into the dentate gyrus of the hippocampus in male rats and early-LTP was induced by weak tetanic stimulation of the medial perforant path. Dorsal right hippocampi were removed, membrane proteins were extracted, separated by blue-native gel electrophoresis with subsequent immunoblotting using brain receptor antibodies. Spatial training resulted into reinforcement of LTP and the reinforced LTP was persistent for 6h. Receptor complex levels containing GluN1 and GluN2A of NMDARs, GluA1 and GluA2 of AMPARs, nAchα7R and the D(1A) dopamine receptor were significantly-elevated in rat hippocampi of animals underwent spatial learning, whilst levels of GluA3 and 5-HT1A receptor containing complexes were significantly reduced. Evidence for complex formation between GluN1 and D(1A) dopamine receptor was provided by antibody shift assay, co-immunoprecipitation and mass spectrometric analysis. Thus our results propose that behavioural stimuli like spatial learning reinforce early LTP into late LTP and this reinforced LTP is accompanied by changes in certain receptor levels in the membrane fraction of the rat hippocampus., (Copyright © 2015. Published by Elsevier B.V.)

Although protein kinases and phosphatases have been reported to be involved in fear memory, information about these signalling molecules in the individual phases of contextual fear conditioning (cFC) is limited. C57BL/6J mice were tested in cFC, sacrificed and hippocampi were used for screening of approximately 800 protein kinases and phosphatases by protein microarrays with subsequent Western blot confirmation of threefold higher or lower hippocampal levels as compared to foot shock controls. Immunoblotting of the protein kinases and phosphatases screened out was carried out by Western blotting. A network of protein kinases and phosphatases was generated (STRING 9.1). Animals learned the task in the paradigm and protein kinase and phosphatase levels were determined in the individual phases acquisition, consolidation and retrieval and compared to foot shock controls. Protein kinases discoidin containing receptor 2 (DDR2), mitogen activated protein kinase kinase kinase 7 (TAK1), protein phosphatases dual specificity protein phosphatase (PTEN) and protein phosphatase 2a (PP2A) were modulated in the individual phases of cFC. Phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase and phosphatidylinositol-3 kinase (PI3K) that is interacting with PTEN were modulated as well. Freezing time was correlating with PP2A levels in the retrieval phase of cFC. The abovementioned protein kinases, phosphatases and inositol-signalling enzymes were not reported so far in cFC and the results are relevant for interpretation of previous and design of future studies in cFC or fear memory. Protein phosphatase PP2A was, however, the only signalling compound tested that was directly linked to retrieval in the cFC., (Copyright © 2014. Published by Elsevier B.V.)

Although the roles of AMPAR (α-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor), NMDAR (N-methyl-D-aspartate receptor) and 5HT1AR (5-hydroxytryptamine sub type 1A) in contextual fear conditioning (cFC) have been studied, information about receptor-containing complexes (RC) is not available. Moreover, there are no data on membrane or endosomal NMDA-, 5HT1A- or AMPA-RC levels, which would likely be indicative of the trafficking of these receptors. cFC was carried out in C57BL/6j mice and animals were sacrificed in the individual phases and hippocampi were taken for the determination of receptor complex and subunit levels using BN- and SDS-PAGE with subsequent Western blotting. GluA1-4, GluN1 (NMDAR subunit NR1)- and 5HT1A-RC were differentially regulated during the individual phases and differentially regulated in the membrane and endosomal fractions. GluA1-RC levels in the membrane were increased in acquisition, consolidation and retrieval phases; GluA2-RC and GluA3-RC membrane levels were reduced and modulated in early endosomes during these phases. GluA4-RC and GluN1-RC levels as well as their subunits showed the same pattern in the membrane during consolidation while 5HT1A-RC membrane and endosome levels were mainly increased during consolidation and retrieval. Taken together, the results suggest that levels of 5-HT1A-RC, NMDA-RC and AMPA-RC and subunits in membrane and endosomal preparations are paralleling individual phases of cFC. The findings from the current study suggest phase-specific receptor complex and subunit formation and propose that receptor complexes should be examined in parallel with receptor subunits to aid the interpretation of previous work and to design future work on neurotransmitter receptors in memory paradigms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The link between the cholinergic and serotonergic system in cognitive function is well-documented. There is, however, limited information on spatial memory and this formed the rationale to carry out a study with the aim to show a specific link between nicotinic and serotonergic receptor complexes rather than the corresponding subunits, to spatial memory retrieval in a land maze. A total of 46 mice were used and divided into two groups, trained and untrained (yoked) in the multiple-T-Maze (MTM) and following training during the first four days, probe trials for memory retrieval were performed on days 8, 16 and 30. Six hours following scarification, hippocampi were taken for the analysis of native receptor complex levels using blue-native gels followed by immunoblotting with specific antibodies. 5-HT1A-, 5-HT7-, nAChα4- and nACh-α7-containing receptor complexes were observed and were paralleling memory retrievals and receptor complex levels were shown to be significantly different between trained and yoked animals. Only levels of a nicotinic acetylcholine α7 receptor-containing complex at an apparent molecular weight of approximately 480kDa were shown to be linked to memory retrieval on day 8 but not to retrievals on days 16 and 30 when memory extinction has taken place. Correlation between nAChα4-, 5-HT1A- and 5-HT7-containing receptors and latencies on day 16 may point to a probable link in extinction mechanisms. A series of the abovementioned receptor complexes were correlating among each other probably indicating a serotonergic/cholinergic network paralleling spatial memory formation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although a large series of reports on monoamine receptor (MAR) biochemistry and pharmacology in aging are available, work on MAR complexes rather than subunits is limited. It was the aim of the study to determine MAR complexes in hippocampi of three different age groups (3-12 and 18 months) in the mouse and to link MAR changes to spatial memory retrieval in the Morris water maze (MWM). MAR complexes were separated by blue native electrophoresis. Immunohistochemistry was performed in order to show the pattern of dopamine receptors and its colocalizations. D1R, D2R and 5-HT7R containing receptor complex levels were decreasing with age while 5-HT1AR-containing complex levels were increasing with age. D1R, 5-HT7R and 5-HT1AR were significantly correlating with the time spent in the target quadrant, representing retrieval in the MWM. D1R and D2R immunoreactivity was decreasing in an area-dependent pattern and D1R and D2R were colocalized. Individual monoamine receptors are linked to spatial memory retrieval and are modulated by age. The findings are relevant for interpretation of previous and design of future work on brain receptors, spatial memory and aging., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Protein phosphorylation is a well-known and well-documented mechanism in memory processes. Although a large series of protein kinases involved in memory processes have been reported, information on phosphoproteins is limited. It was therefore the aim of the study to determine a partial and differential phosphoproteome along with the corresponding network in hippocampus of a wild caught mouse strain with excellent performance in several paradigms of spatial memory. Apodemus sylvaticus mice were trained in the Barnes maze, a non-invasive test system for spatial memory and untrained mice served as controls. Animals were sacrificed 6h following memory retrieval, hippocampi were taken, proteins extracted and in-solution digestion was carried out with subsequent iTRAQ double labelling. Phosphopeptides were enriched by a TiO2-based method and semi-quantified using two fragmentation principles on the LTQ-orbitrap Velos. In hippocampi of trained animals phosphopeptide levels representing signalling, neuronal, synaptosomal, cytoskeletal and metabolism proteins were at least twofold reduced or increased. Furthermore, a network revealing a link to pathways of ubiquitination, the androgen receptor, small GTPase Rab5 and MAPK signaling as well as synucleins was constructed. This work is relevant for interpretation of previous work and the design of future studies on protein phosphorylation in spatial memory., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although a few individual members of the protein kinase C (PKC) family were studied in spatial memory no systematic approach was carried out to concomitantly determine all described PKC family members in spatial memory of the mouse. It was therefore the aim of the current study to link hippocampal PKCs to memory retrieval in the Morris water maze (MWM). CD1 mice were trained (n=9) or untrained (n=9) in the MWM, hippocampi were taken 6h following the test for memory retrieval and PKCs were determined in mouse hippocampi by immunoblotting. The trained animals learned the spatial memory task and kept memory at the probe trial. PKCs alpha and epsilon were comparable between groups while PKCs beta, delta, gamma (two forms, i.e. two bands on Western blotting), zeta (2 forms) were higher in trained mice and theta (2 forms) were lower in trained mice. PKC gamma (1 form) was significantly correlating with the time spent in the target quadrant (r=0.7933; P=0.0188). Changes of hippocampal levels of PKCs beta, delta, gamma, zeta and theta were paralleling memory retrieval of the MWM task but correlations revealed that spatial memory retrieval was only linked to one form of PKC gamma. Results are also in agreement with a recent publication showing that PKM zeta is not required for memory formation. These findings may be relevant for the interpretation of previous work and the design of future work on the protein kinase C family in spatial memory of the mouse., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although a series of amino acid analogs have been shown to modulate brain function, information on the pharmacology of alpha-alkylated amino acids (AAAA) is limited. In particular there is no information on the effect of these amino acid analogs (AAA) on the elevated plus maze, the tail suspension test and the forced swim test. It was therefore the aim of the study to test a series of AAAA in these paradigms in order to explore behavioral activities of this compound class. 10 male mice per group aged between 10 and 14 weeks were used. Vehicle-treated controls were used in addition to intraperitoneal injections of 1, 10 and 100mg/kg body weight of each, alpha-amino-isobutyic acid (AIB), isovaline (IVA), alpha-propyl-alanine (APA), alpha-butyl-alanine (ABA), alpha-pentyl-alanine (APnA), alpha-ethylphenylglycine (AEPG) and alpha-methyl-valine (AMV). The elevated plus maze (EPM), the tail suspension test (TST) and forced swim test (FST) were used for behavioral testing. There were dose-dependent results: all compounds increased time and pathlength in the open arm of the EPM at least at one dose administered. In the TST and in the FST only the 100mg dose was showing an effect. The results show pharmacological activity modifying the EPM in low doses suggesting the use in treatment of behavioral traits and symptoms represented by or linked to the EPM including anxiety-related behavior including depression. Compounds acting at higher doses may be used to induce behavioral changes and thus serve as neurobiological-neuropharmacological tools., (Copyright © 2013. Published by Elsevier B.V.)

Although a series of amino acids (AA) have been associated with spatial memory formation, there is limited information on concentrations of beta-alanine and citrulline in rodent brains. Given the importance of AA metabolism in cognitive functions it was the aim of the study to determine hippocampal levels of beta-alanine and citrulline in rats during two different phases of memory retrieval in a spatial memory paradigm. Ten rats were used per group and the first group was trained and sacrificed five min, the second six hours following retrieval in the Morris Water Maze (MWM) and the third and fourth group were untrained, yoked controls. Hippocampi were taken and free AA were determined using a well-established HPLC protocol. Beta-alanine and citrulline levels were higher in trained rat hippocampi, during both, early and late phase of memory retrieval. Taurine, methionine, cysteine, lysine and ornithine levels were higher in yoked rats at the late phase while tyrosine was higher in yoked rats during the early phase. There were no significant correlations between time spent in the target quadrant and any of the AA levels. Herein, an AA pattern, different between yoked and trained animals at early and late phase of memory retrieval is shown, indicating probable involvement of different AA pathways in animals trained and untrained in the MWM. The results may be useful for the interpretation of previous studies and the design of future experiments to identify amino acids as possible targets for modulating spatial memory., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although the involvement of the mTOR (mammalian target of rapamycin) system in memory processes has been reported, information on the effect of rapamycin on spatial learning and memory is limited. It was therefore the aim of the study to show the effect of parenteral rapamycin administration to C57BL/6J mice on performance in the multiple T-maze (MTM) and to determine hippocampal mTOR activity. Rapamycin-treated and -untreated/trained/probed mice are the main part of the experiment considering retrieval and acquisition or consolidation of spatial memory. Six hours following euthanasia hippocampi were extirpated and used for evaluation of mTOR activity as represented by hippocampal levels of S6 protein and its phosphorylated active form (phospho S6 protein, S240,244), a read out of mTOR complex 1 activity. Mice given i.p. rapamycin learned the task of the MTM but failed at the probe trial, showing absence of the phosphorylated active form of S6 protein, indicating inhibition of mTOR activity. Herein, impairing effects of rapamycin on retrieval but not on acquisition or consolidation of spatial memory are shown. Deficient memory retrieval was paralleled by inhibition of mTOR complex 1 activity. The current study extends knowledge on rapamycin in memory mechanisms and challenges work on deeper insights into the role of mTOR in different phases of memory formation and retrieval., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The N-methyl-d-aspartic acid receptor (NMDAR) is a well-documented key element in the formation of several memories including spatial, olfactory and contextual memory. Although receptor subunits have been linked to memory formation, data on the involvement of the NMDAR complexes is limited. In previous work CD1 mice were trained in the Barnes maze, a low-stress landmaze, and yoked controls were serving as controls. Hippocampal samples from this behavioural study were taken for comparing NMDAR complexes. Hippocampi were taken and stored until analysis at -80 °C. Membrane proteins were extracted from hippocampi using an ultracentrifugation step and applied on Blue Native gels that in turn were used for immunoblotting with antibodies against subunits NR1, NR2A and NR2B. The subunit content of the complexes was determined by denaturing two-dimensional gel electrophoresis and subsequent immunoblotting. An NMDAR complex with an apparent molecular weight between between 146 and 242 kDa, probably representing an NR1 dimer was the only complex that was significantly different between trained and yoked animals. A series of NMDAR complexes containing modulatory subunits NR2A or NR2B or both were detected. All complexes contained the NR1 subunit. The NR1 dimer complex level, increased in memory formation, may be directly or indirectly involved in the process of spatial memory formation in the CD1 mouse. The results are enabling and challenging further NMDAR studies, both, at the pharmacological and molecular level. Moreover, several NMDAR complexes in the CD1 mouse were shown to be mainly heteropolymers of subunits NR1, NR2A and NR2B, although other recently described subunits were not tested due to unavailability of specific antibodies. Determination of native receptor complexes rather than individual subunits is mandatory and provides the molecular basis for understanding mechanisms of spatial memory., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although the involvement of the serotonin 1A receptor (5-HT(1A) R) in memory has been shown by several groups there is no information about the 5-HT(1A) R complex but rather the monomeric form. Moreover, there is insufficient information on the characterization of the antigen, the 5-HT(1A) R. PWD/PhJ mice, a wild-caught strain that was inbred at The Jackson Laboratories were used for the experiments. The Barnes maze (BM) paradigm for the evaluation of spatial memory was chosen because of experience with this setting in our laboratory. Mice were sacrificed 6 h following the probe trial on day 12, hippocampi were extirpated, proteins extracted and run on blue native gels with subsequent immunoblotting with a specific antibody against the mouse 5-HT(1A) R. Densitometry of the single band from the immunoblots was carried out. The receptor from the complex was identified by mass spectrometry (nano-LC-ESI-MS/MS). A serotonin receptor 1A complex was identified by immunoblotting at the apparent molecular weight of 480 kDa indicating the presence of a homopolymer as denaturation only revealed a single band at approx. 50 kDa. 5-HT(1A) R levels were significantly higher in the trained group as compared to yoked controls. The hippocampal 5-HT(1A) R of the trained group was unambiguously identified. Taken together, a serotonin receptor 1A homopolymer is associated with memory training effects in the BM using PWD/PhJ mice. This observation shows that a specific complex rather than a receptor subunit as previously shown is involved in memory process and the receptor protein was characterized., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Evaluation of spatial learning and memory is mainly carried out using the Morris water maze as a single paradigm. We intended to test whether mice in the Barnes maze and Multiple T-maze would lead to comparable results and to test two individual mouse strains with different anxiety levels. C57BL/6J and CD1 male mice were used in the experiments. During the acquisition phase, learning was measured using parameters latency, path length, errors in the BM and correct decisions in MTM. Mice were trained for 4 days and probe trials were performed on days 5 and 12. Latencies reduction over the training period indicated that both strains learned all tasks. During retention phase at days 5 and 12 C57BL/6J performed the Barnes maze and Multiple T-maze task better than CD1 mice while CD1 performed better than C57BL/6J in the Morris water maze. In the BM at day 12, C57BL/6J kept the level of visits to target observed at day 5 whereas CD1 performed worse. Strain- and task-dependent differences were observed using the three mazes. Therefore, fair evaluation of spatial memory demands application of (at least) two different test systems, a water- and a land maze. Different anxiety-related behaviour as well as stress-responses in the strains used may help to interpret the findings reported and again may propose the use of at least two mouse strains when robust evaluation of spatial memory is considered.

SGS742 has been reported to increase spatial memory in rodents. However, effects of SGS742 have not been systematically assessed in different strains so far and indeed strains show different cognitive abilities per se. The aim of the study was therefore to examine the effect of SGS742 in three different inbred (C57BL/6J, DBA/2, BALB/c) strains and three outbred strains (CD1, CF1, OF1). Mice were administered intra-peritonial (3-aminopropyl)n-butylphosphinic acid (SGS742; GABA (B)-receptor antagonist) and tested in the Morris water maze (MWM). Open field, elevated plus maze, neurological observational battery and rota rod were carried out to support interpretation of data. SGS742 enhanced performance in the MWM in an inbred strain, C57BL/6J and in the outbred strain OF1 in terms of learning and memory formation at the consolidation level. A series of side effects as e.g. reduced motor coordination and proprioception were noticed that, however, may not have been influencing results observed in the cognition task. In conclusion, SGS742 enhanced cognitive performance in two mouse strains and we learned that testing compounds for effects on spatial memory should be carried out in several strains or even different species in order to claim cognitive enhancement.

Inbred mouse strains have different genetic backgrounds that can result in impairment of synaptic plasticity and memory. Strain-dependent performance in behavioral and cognitive tasks is well-documented. Hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that may underlie some forms of learning and memory has been shown to differ significantly between inbred mouse strains. However, an effect of strain on the expression of proteins, critically involved in synaptic plasticity, learning and memory has not been described yet. We have been addressing this question by determining expressional levels of a panel of proteins involved in neuronal information processing in hippocampus of five mouse strains by immunoblotting. Four inbred strains (FVB/N, C57Bl/6J, 129S2/Sv and Balb/c), commonly used for generating genetically modified mice and for conventional experiments in pharmacology and toxicology and one outbred strain (OF1) have been selected. A significant effect of strain was detected for total and phosphorylated calcium-calmodulin dependent kinase IIalpha (CaMKII, pCaMKII), phosphorylated mitogen-activated protein kinase (pMAPK), total and phosphorylated calcium-responsive element binding 1 (creb, pcreb), early-growth response protein 1 (egr 1), brain derived neurotrophic factor (BDNF), drebrin and postsynaptic density-95 (PSD-95). These results may indicate genetic determination of synaptic plasticity-related mechanisms relevant for the molecular events mediating hippocampal information processing and storage. Data presented herein highlight the importance of careful selection of the mouse strain for studies of synaptic plasticity.

Several protein cascades are proposed to be involved in the formation of synaptic plasticity and have been linked to neuronal information processing and storage. Although modified expression of specific proteins following behavioral testing has been shown, no systematic approach for their concomitant determination has been reported. We therefore determined hippocampal expression of signaling proteins, transcription factors and synaptosomal-associated proteins representing key elements of neuronal plasticity in mice following behavioral training. Male FVB/N mice, 12 weeks of age, were used for behavioral testing. After completion of tests mice were sacrificed and hippocampi were dissected. Levels of total and autophosphorylated (T286) alphacalcium-calmodulin dependent kinase II (CaMKII, pCaMKII), total and phosphorylated mitogen-activated protein kinase (MAPK, pMAPK), total and phosphorylated calcium-responsive element binding (creb, pcreb), early-growth response protein 1 (egr-1), brain derived neurotrophic factor (BDNF), tyrosine kinase receptor B (trk B), drebrin and postsynaptic density-95 (PSD-95) were quantified in hippocampi of behavior trained animals (n=7) and naïve caged controls (n=7). Expression of pCaMKII, BDNF, PSD-95 and egr-1 was significantly increased in the behavior-trained group. Expression of total CaMKII, total and pMAPK, total and pcreb, trk B and drebrin was comparable between groups. Detection of significantly increased pCaMKII, BDNF, PSD-95 and egr-1 induced by behavioral training at the protein level per se is intriguing and supports the proposed importance of these molecules for neuronal information storage.

There is evidence that ageing both in humans and animals is accompanied by changes in emotional behaviour. Behavioural studies in rats point to an increase in emotional reactivity and/or anxiety-related behaviour with age. Here we studied social interaction in young adult (3 months) and aged (30 months old) rats using an established test system for anxiety-related behaviour. Using Fos expression as a marker of neuronal activation, we aimed to investigate whether age-related differences in anxiety would be reflected by changes in neuronal activity in brain regions known to be sensitive to fear- and anxiety-related stimuli. Aged rats spent significantly less time (75%) in active social interaction than young rats, without concomitant changes in general locomotor activity. Social interaction enhanced Fos expression both in young and aged rats in several anxiety-related brain areas. Lower Fos response in aged versus young rats was noted in the dorsomedial, dorsolateral and ventrolateral part of the periaqueductal grey, the medial and basolateral amygdala and parvocellular region of the paraventricular hypothalamic nucleus, while no differences in Fos expression were observed in the other regions examined, including the hippocampus, septum or locus coeruleus. These results demonstrate age-related reduction in social interaction, indicative of enhanced anxiety-related behaviour in aged rats. However, since the supposedly increased anxiety level was not accompanied by augmented Fos expression in any of the key brain areas of the fear/anxiety circuitry known to be activated by anxiogenic stimuli, it is suggested that reduced social interaction does not reflect enhanced anxiety in aged rats., (Copyright 2003 Elsevier B.V.)