Volker Korz

Hippocampal Testosterone Relates to Reference Memory Performance and Synaptic Plasticity in Male Rats

Steroids are important neuromodulators influencing cognitive performance and synaptic plasticity. While the majority of literature concerns adrenal- and gonadectomized animals, very little is known about the “natural” endogenous release of hormones during learning. Therefore, we measured blood and brain (hippocampus, prefrontal cortex) testosterone, estradiol, and corticosterone concentrations of intact male rats undergoing a spatial learning paradigm which is known to reinforce hippocampal plasticity. We found significant modulations of all investigated hormones over the training course. Corticosterone and testosterone were correlated manifold with behavior, while estradiol expressed fewer correlations. In the recall session, testosterone was tightly coupled to reference memory (RM) performance, which is crucial for reinforcement of synaptic plasticity in the dentate gyrus. Intriguingly, prefrontal cortex and hippocampal levels related differentially to RM performance. Correlations of testosterone and corticosterone switched from unspecific activity to specific cognitive functions over training. Correspondingly, exogenous application of testosterone revealed different effects on synaptic and neuronal plasticity in trained versus untrained animals. While hippocampal long-term potentiation (LTP) of the field excitatory postsynaptic potential (fEPSP) was prolonged in untrained rats, both the fEPSP- and the population spike amplitude (PSA)-LTP was impaired in trained rats. Behavioral performance was unaffected, but correlations of hippocampal field potentials with behavior were decoupled in treated rats. The data provide important evidence that besides adrenal, also gonadal steroids play a mechanistic role in linking synaptic plasticity to cognitive performance.

Stress induced hippocampal mineralocorticoid and estrogen receptor β gene expression and long-term potentiation in male adult rats is sensitive to early-life stress experience.

Glucocorticoid hormones and their receptors have been identified to be involved in emotional and cognitive disorders in early stressed subjects during adulthood. However, the impact of other steroid hormones and receptors has been considered less. Especially, functional roles of estrogen and estrogen receptors in male subjects are largely unknown. Therefore, we measured hippocampal concentrations of 17β-estradiol, corticosterone and testosterone, as well as the gene expression of estrogen receptor α and β (ERα, β), androgen receptor (AR), glucocorticoid (GR) and mineralocorticoid (MR) receptors after stress in adulthood in maternally separated (MS+; at postnatal days 14-16 for 6h each day) and control (MS-) male rats. In vivo hippocampal long-term potentiation (LTP) serves as a cellular model of learning and memory formation. Population spike- (PSA) and the fEPSP-LTP within the dentate gyrus (DG) were reinforced by elevated-platform-stress (EP-stress) in MS- but not in MS+ rats. MR- and ERβ-mRNA were upregulated 1h after EP-stress in MS- but not in MS+ rats as compared to non-stressed littermates. Infusion of an MR antagonist before LTP induction blocked early- and late-PSA- and -fEPSP-LTP, whereas blockade of ERβ impaired only the late PSA-LTP. Application of a DNA methyltransferase (DNMT) inhibitor partly restored the LTP-reinforcement in MS+ rats, accompanied by a retrieval of ERβ- but not MR-mRNA upregulation. Basal ERβ gene promoter methylation was similar between groups, whereas MS+ and MS- rats showed different methylation patterns across CpG sites after EP-stress. These findings indicate a key role of ERβ in early-stress mediated emotionality and emotion-induced late-LTP in adult male rats via DNA methylation mechanisms.

Hippocampal receptor complexes paralleling LTP reinforcement in the spatial memory holeboard test in the rat

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.

Estrogen Receptor α Functions in the Regulation of Motivation and Spatial Cognition in Young Male Rats

Estrogenic functions in regulating behavioral states such as motivation, mood, anxiety, and cognition are relatively well documented in female humans and animals. In males, however, although the entire enzymatic machinery for producing estradiol and the corresponding receptors are present, estrogenic functions have been largely neglected. Therefore, and as a follow-up study to previous research, we sub-chronically applied a specific estrogen receptor α (ERα) antagonist in young male rats before and during a spatial learning task (holeboard). The male rats showed a dose-dependent increase in motivational, but not cognitive, behavior. The expression of hippocampal steroid receptor genes, such as glucocorticoid (GR), mineralocorticoid (MR), androgen (AR), and the estrogen receptor ERα but not ERβ was dose-dependently reduced. The expression of the aromatase but not the brain-derived neurotrophic factor (BDNF) encoding gene was also suppressed. Reduced gene expression and increased behavioral performance converged at an antagonist concentration of 7.4 µmol. The hippocampal and blood serum hormone levels (corticosterone, testosterone, and 17β-estradiol) did not differ between the experimental groups and controls. We conclude that steroid receptors (and BDNF) act in a concerted, network-like manner to affect behavior and mutual gene expression. Therefore, the isolated view on single receptor types is probably insufficient to explain steroid effects on behavior. The steroid network may keep motivation in homeostasis by supporting and constraining the behavioral expression of motivation.

Repeated application of Modafinil and Levodopa reveals a drug-independent precise timing of spatial working memory modulation.

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.

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.

Differential effects of wake promoting drug modafinil in aversive learning paradigms

Modafinil (MO) an inhibitor of the dopamine transporter was initially approved to treat narcolepsy, a sleep related disorder in humans. One interesting “side-effect” of this drug, which emerged from preclinical and clinical studies, is the facilitation of cognitive performance. So far, this was primarily shown in appetitive learning paradigms, but it is yet unclear whether MO exerts a more general cognitive enhancement effect. Thus, the aim of the present study in rats was to extend these findings by testing the effects of MO in two aversive paradigms, Pavlovian fear conditioning (FC) and the operant two-way active avoidance (TWA) learning paradigms. We discovered a differential, task-dependent effect of MO. In the FC paradigm MO treated rats showed a dose-dependent enhancement of fear memory compared to vehicle treated rats, indicated by increased context-related freezing. Cue related fear memory remained unaffected. In the TWA paradigm MO induced a significant decrease of avoidance responses compared to vehicle treated animals, while the number of escape reactions during the acquisition of the TWA task remained unaffected. These findings expand the knowledge in the regulation of cognitive abilities and may contribute to the understanding of the contraindicative effects of MO in anxiety related mental disorders.

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.

Dorsal hippocampal brain receptor complexes linked to the protein synthesis-dependent late phase (LTP) in the rat

In order to link major brain receptor complex levels to in vivo electrically induced LTP, a bipolar stimulation electrode was chronically implanted into the perforant path, while two monopolar recording electrodes were implanted into the dentate gyrus of the dorsal hippocampus. The recording electrode was measuring extracellular excitatory postsynaptic potentials, while the other one measured population spikes. Immunoblotting of native receptor proteins was carried out in the DH based upon blue-native gel electrophoresis and immunoprecipitation followed by mass spectrometrical identification of the NR1-GluA1-GluA2 complex was used to provide evidence for complex formation. The induction of LTP in DH was proven and NMDA receptor complex levels containing NR1, GluA1, GluA2 and GluA3 were modulated by LTP induction. The LTP-associated changes of receptor complex levels may indicate concerted action, interaction and represent a pattern of major brain receptor complexes in the DH following electrical induction of LTP in the rat.