Yogesh D. Aher

Spatial and Working Memory Is Linked to Spine Density and Mushroom Spines.

Background Changes in synaptic structure and efficacy including dendritic spine number and morphology have been shown to underlie neuronal activity and size. Moreover, the shapes of individual dendritic spines were proposed to correlate with their capacity for structural change. Spine numbers and morphology were reported to parallel memory formation in the rat using a water maze but, so far, there is no information on spine counts or shape in the radial arm maze (RAM), a frequently used paradigm for the evaluation of complex memory formation in the rodent. Methods 24 male Sprague-Dawley rats were divided into three groups, 8 were trained, 8 remained untrained in the RAM and 8 rats served as cage controls. Dendritic spine numbers and individual spine forms were counted in CA1, CA3 areas and dentate gyrus of hippocampus using a DIL dye method with subsequent quantification by the Neuronstudio software and the image J program. Results Working memory errors (WME) and latency in the RAM were decreased along the training period indicating that animals performed the task. Total spine density was significantly increased following training in the RAM as compared to untrained rats and cage controls. The number of mushroom spines was significantly increased in the trained as compared to untrained and cage controls. Negative significant correlations between spine density and WME were observed in CA1 basal dendrites and in CA3 apical and basal dendrites. In addition, there was a significant negative correlation between spine density and latency in CA3 basal dendrites. Conclusion The study shows that spine numbers are significantly increased in the trained group, an observation that may suggest the use of this method representing a morphological parameter for memory formation studies in the RAM. Herein, correlations between WME and latency in the RAM and spine density revealed a link between spine numbers and performance in the RAM.

The effect of modafinil on the rat dopamine transporter and dopamine receptors D1–D3 paralleling cognitive enhancement in the radial arm maze

A series of drugs have been reported to increase memory performance modulating the dopaminergic system and herein modafinil was tested for its working memory (WM) enhancing properties. Reuptake inhibition of dopamine, serotonin (SERT) and norepinephrine (NET) by modafinil was tested. Sixty male Sprague–Dawley rats were divided into six groups (modafinil-treated 1–5–10 mg/kg body weight, trained and untrained and vehicle treated trained and untrained rats; daily injected intraperitoneally for a period of 10 days) and tested in a radial arm maze (RAM), a paradigm for testing spatial WM. Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT-CC and pDAT-CC) and complexes containing the D1–3 dopamine receptor subunits (D1–D3-CC) were determined. Modafinil was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 11.11 μM; SERT 1547 μM; NET 182 μM). From day 8 (day 9 for 1 mg/kg body weight) modafinil was decreasing WM errors (WMEs) in the RAM significantly and remarkably at all doses tested as compared to the vehicle controls. WMEs were linked to the D2R-CC and the pDAT-CC. pDAT and D1–D3-CC levels were modulated significantly and modafinil was shown to enhance spatial WM in the rat in a well-documented paradigm at all the three doses and dopamine reuptake inhibition with subsequent modulation of D1–3-CC is proposed as a possible mechanism of action.

A Novel Heterocyclic Compound CE-104 Enhances Spatial Working Memory in the Radial Arm Maze in Rats and Modulates the Dopaminergic System.

Various psychostimulants targeting monoamine neurotransmitter transporters (MATs) have been shown to rescue cognition in patients with neurological disorders and improve cognitive abilities in healthy subjects at low doses. Here, we examined the effects upon cognition of a chemically synthesized novel MAT inhibiting compound 2-(benzhydrylsulfinylmethyl)-4-methylthiazole (named as CE-104). The efficacy of CE-104 in blocking MAT [dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter] was determined using in vitro neurotransmitter uptake assay. The effect of the drug at low doses (1 and 10 mg/kg) on spatial memory was studied in male rats in the radial arm maze (RAM). Furthermore, the dopamine receptor and transporter complex levels of frontal cortex (FC) tissue of trained and untrained animals treated either with the drug or vehicle were quantified on blue native PAGE (BN-PAGE). The drug inhibited dopamine (IC50: 27.88 μM) and norepinephrine uptake (IC50: 160.40 μM), but had a negligible effect on SERT. In the RAM, both drug-dose groups improved spatial working memory during the performance phase of RAM as compared to vehicle. BN-PAGE Western blot quantification of dopamine receptor and transporter complexes revealed that D1, D2, D3, and DAT complexes were modulated due to training and by drug effects. The drug’s ability to block DAT and its influence on DAT and receptor complex levels in the FC is proposed as a possible mechanism for the observed learning and memory enhancement in the RAM.

Frontal cortex and hippocampus neurotransmitter receptor complex level parallels spatial memory performance in the radial arm maze.

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.

A novel heterocyclic compound targeting the dopamine transporter improves performance in the radial arm maze and modulates dopamine receptors D1-D3

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.

R-Modafinil exerts weak effects on spatial memory acquisition and dentate gyrus synaptic plasticity

Modafinil is a wake promoting drug approved for clinical use and also has cognitive enhancing properties. Its enantiomer R-Modafinil (R-MO) is not well studied in regard to cognitive enhancing properties. Hence we studied its effect in a spatial memory paradigm and its possible effects on dentate gyrus long-term potentiation (DG-LTP). Clinically relevant doses of R-MO, vehicle dimethyl sulfoxide (DMSO) or saline were administered for three days during the hole-board test and in in vivo DG-LTP. Synaptic levels of dopamine receptors D1R, D2R, dopamine transporter (DAT), and its phosphorylated form (ph-DAT) in DG tissue 4 h after LTP induction were quantified by western blot analysis. Monoamine reuptake and release assays were performed by using transfected HEK-293 cells. Possible neurotoxic side effects on general behaviour were also studied. R-MO at both doses significantly enhanced spatial reference memory during the last training session and during memory retrieval compared to DMSO vehicle but not when compared to saline treated rats. Similarly, R-MO rescues DG-LTP from impairing effects of DMSO. DMSO reduced memory performance and LTP magnitude when compared to saline treated groups. The synaptic DR1 levels in R-MO groups were significantly decreased compared to DMSO group but were comparable with saline treated animals. We found no effect of R-MO in neurotoxicity tests. Thus, our results support the notion that LTP-like synaptic plasticity processes could be one of the factors contributing to the cognitive enhancing effects of spatial memory traces. D1R may play an important regulatory role in these processes.

Individual Differences in Male Rats in a Behavioral Test Battery: A Multivariate Statistical Approach

Animal models for anxiety, depressive-like and cognitive diseases or aging often involve testing of subjects in behavioral test batteries. The large number of test variables with different mean variations and within and between test correlations often constitute a significant problem in determining essential variables to assess behavioral patterns and their variation in individual animals as well as appropriate statistical treatment. Therefore, we applied a multivariate approach (principal component analysis) to analyse the behavioral data of 162 male adult Sprague-Dawley rats that underwent a behavioral test battery including commonly used tests for spatial learning and memory (holeboard) and different behavioral patterns (open field, elevated plus maze, forced swim test) as well as for motor abilities (Rota rod). The high dimensional behavioral results were reduced to fewer components associated with spatial cognition, general activity, anxiety-, and depression-like behavior and motor ability. The loading scores of individual rats on these different components allow an assessment and the distribution of individual features in a population of animals. The reduced number of components can be used also for statistical calculations like appropriate sample sizes for valid discriminations between experimental groups, which otherwise have to be done on each variable. Because the animals were intact, untreated and experimentally naïve the results reflect trait patterns of behavior and thus individuality. The distribution of animals with high or low levels of anxiety, depressive-like behavior, general activity and cognitive features in a local population provides information of the probability of their appeareance in experimental samples and thus may help to avoid biases. However, such an analysis initially requires a large cohort of animals in order to gain a valid assessment.

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

HIGHLIGHTS5‐((Benzhydrylsulfinyl)methyl) thiazole (CE‐123) was synthetized as a novel dopamine reuptake inhibitor.CE‐123 with respect to NET, DAT and SERT is a highly selective DAT inhibitor with no basic neurotoxicity.CE‐123 enters the brain and shows lower elimination than R‐modafinil.CE‐123 improves spatial memory performance in the rat. ABSTRACT 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&mgr;M) 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.

A novel heterocyclic compound improves working memory in the radial arm maze and modulates the dopamine receptor D1R in frontal cortex of the Sprague-Dawley rat

&NA; 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 ± 0,8 &mgr;M) 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 10 mg/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 1 mg/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. HighlightsCE‐125 is non‐neurotoxic compound and specific DAT reuptake inhibitor.CE‐125 improvesspatial memoryperformance and increases the WMI in aradial arm maze.CE‐125 modulates the dopamine receptor D1R.