Sergiu Dalm

Age-Related Changes in Hypothalamic-Pituitary-Adrenal Axis Activity of Male C57BL/6J Mice

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.

Non-invasive stress-free application of glucocorticoid ligands in mice

Most drug delivery procedures induce stress, which might interfere with the pharmacological action of the drug and behaviour. Stress is deduced from high and long-lasting elevations of the hormone corticosterone. We set out to develop a non-invasive, stress-free method of drug delivery in mice. Validation consisted of delivery of glucocorticoid ligands via oats to male C57BL/6J mice. Oat consumption induced a small increase in corticosterone concentrations after 15 min (<50 ng/ml) that returned to low resting levels at t=30 (<10 ng/ml). Gavage and intraperitoneal (i.p.) vehicle injections resulted in long-lasting corticosterone elevations (>100 ng/ml at t=30 and approximately 50 ng/ml at t=60 min after delivery). Adding corticosterone to oats resulted in threefold higher plasma corticosterone in the 15.0-mg/kg group (+/-250 ng/ml) compared to the 4.5-mg/kg group at t=30 and 90. Application of the glucocorticoid receptor antagonist RU38486 (200 mg/kg) elevated plasma corticosterone for at least 8h. Additional swimming increased corticosterone even further. Presumably, already the small oat-consumption-induced increase of corticosterone requires negative feedback via glucocorticoid receptors. In conclusion, the context-dependent and dose-controlled application of drugs via oats avoids confounding strong stress system activation and makes it suitable for studies on learning and memory processes.

Differential effect of corticosterone on spatial learning abilities in apolipoprotein E knockout and C57BL/6J mice

Previously, we found that repeated exposure to predator stress corrected the deficit in spatial learning of apolipoprotein E-knockout (apoE0/0) mice, but impaired cognitive performance of wild-type mice. Here we show that elevated corticosterone concentrations, accomplished by subcutaneously implanted pellets, results in similar genotype-related effects on water maze learning: while apoE0/0 mice improved their spatial learning abilities, wild-type mice (C57/Bl6J) became impaired. These results suggest that corticosterone mediates the lasting effects of environmental challenges on apoE-genotype related cognitive performance.

Repeated rat exposure inhibits the circadian activity patterns of C57BL/6J mice in the home cage.

Exposing male C57BL/6J mice repeatedly, in an unpredictable and uncontrollable fashion to rats, alters their cognitive performance and the neuroendocrine stress response, weeks to months after the rat stress. Continuous observation of the behavioural activity of male C57BL/6J mice in their home cage before (baseline) and after rat exposure could reveal if repeated rat exposure leads to changes in circadian activity patterns, which is a key feature of chronic stress and stress-related disorders in humans. Rat stress (1) decreased exploratory and foraging activity as characterized by increased time spent in the shelter and less time in the open area; (2) reduced sucrose consumption and inhibited the development of sucrose preference, suggesting changes in the reward system and (3) the exploration pattern in a novel environment included more behavioural perseverations, but no change in general locomotor activity. Comparison to baseline activity pattern, i.e., before any intervention, revealed that already the control procedure to rat exposure (spending the same amount of time in another cage) disrupted the organization of behavioural activity patterns, albeit to a different and lesser degree than observed in rat stressed mice. While only the longitudinal design of the study allowed detecting these dynamic patterns of circadian activities, the distinct behavioural changes in foraging and explorative activities support our notion that repeated rat exposure might serve as mouse model of chronic stress.

Increased corticosterone secretion and early-onset of cognitive decline in female apolipoprotein E-knockout mice

In the present study, the interaction of age and apolipoprotein E (apoE)-genetic background on cognitive abilities was investigated in young (5-6 months) and aged (14-16 months) female apolipoprotein E-knockout (apoE0/0) and wild-type mice. Cognitive abilities are known to be affected by the steroid hormones corticosterone and estrogen. Therefore, we measured the activity and reactivity of the hypothalamic-pituitary-adrenal (HPA) axis expressed by circadian corticosterone concentrations and responses to novelty and controlled the regularity of the estrous cycle. Young female apoE0/0 mice acquired the water maze task and showed a similar latency and search strategy to locate the platform as young female wild-type mice. Similar corticosterone responses to novelty were observed in both genotypes. Regularity of the estrous cycle was disturbed in a small percentage of the young apoE0/0 female mice. However, in aged female apoE0/0 mice water maze performance was impaired with search strategies less persistent than in aged wild-type mice. In parallel, increased corticosterone concentrations were measured in apoE0/0 mice in response to novelty and during the circadian cycle. The percentage of mice with an irregular estrous cycle increased with age, but was comparable for apoE0/0 and wild-type mice. Thus, although disruption of the apoE gene affects the regularity of the estrous cycle in young mice, it is the enhanced corticosterone secretion, which parallels the cognitive decline in the aging female apoE0/0 mice.

Stress alleviates reduced expression of cell adhesion molecules (NCAM, L1), and deficits in learning and corticosterone regulation of apolipoprotein E knockout mice

Cell adhesion molecules (CAMs) involved in synaptic changes underlying learning and memory processes, are implicated in the effect of stress on behavioural performance. The present study was designed to test the hypothesis that (i) expression of CAMs is apolipoprotein E‐ (apoE) genotype dependent and (ii) repeated exposure to stress modulates the synthesis of CAMs in an apoE‐genotype dependent manner. Using ELISA we tested this hypothesis and measured expression of NCAM and L1 in different brain regions of naïve and stressed apolipoprotein E‐knockout (apoE0/0) and C57Bl6 (wild‐type) mice. Naïve apoE0/0 mice had elevated basal morning corticosterone and ACTH concentrations and decreased expression of NCAM and L1 compared to wild‐type mice. Repeated exposure of mice to rats, as the common stressor, alleviated the reduction in expression of CAMs in apoE0/0 mice; seven days after the last rat exposure, expression of NCAM was increased in frontal brain and hippocampus whereas expression of L1 was increased in hippocampus and cerebellum. Rat stress attenuated the elevation of basal morning corticosterone concentration in apoE0/0 mice towards concentrations detected in wild‐type mice. Moreover, rat stress improved learning and memory of apoE0/0 mice in the water maze. In conclusion, repeated exposure to stress eliminated apoE‐genotype‐related differences in expression of CAMs. Under these same conditions the differences in cognitive performance and corticosterone concentrations were abolished between wild type and apoE0/0 mice.

Post-training self administration of sugar facilitates cognitive performance of male C57BL/6J mice in two spatial learning tasks.

Spatial memory can be strengthened by adverse stimuli that activate the stress system, and administration of the stress hormone corticosterone in close-context with the learning task. Less is known about modulation of spatial memory by post-training positive reinforcers (reward). Cognitive performance was assessed in male C57BL/6J mice using two learning tasks: the water maze (WM) and circular hole board (CHB). Sugar was chosen as a post-training reinforcer. We expected that the free access to sugar immediately (0 h) after training would facilitate spatial memory; delayed access to sugar (4h after training) or no sugar served as controls. In both tasks, 0 h sugar mice showed superior performance, indicated by shorter latencies and distances to the trained spatial location. The memory facilitating effect of sugar became visible at distinct times during training: on the CHB from the first trial onwards, in the WM on training days 4 and 5. Sugar-rewarded mice kept their superior performance during the free exploration/swim trial, expressed by more persistent search strategies for the exit hole or platform. Post-training sugar reward in close-context with performance strengthens memory via modulation of consolidation. This finding supports the integrative theory of reinforcement and memory. We suggest that our experimental set-up will allow to differentiate between direct effects on memory and alterations in reward processes in animal models of stress-related diseases.

Post-training reward partially restores chronic stress induced effects in mice.

Reduced responsiveness to positive stimuli is a core symptom of depression, known as anhedonia. In the present study, we assessed the expression of anhedonia in our chronic stress mouse model using a subset of read-out parameters. In line with this, we investigated in how far chronic stress would affect the facilitating effect of post-training self-administration of sugar, as we previously observed in naïve mice. Male C57BL/6J mice were repeatedly and at unpredictable times exposed to rats (no physical contact) over the course of two weeks. Following novelty exploration, (non-) spatial learning and memory processes with and without post-training sugar acting as reinforcer, emotionality, reward sensitivity and corticosterone levels were determined. We found that (1) the effects of chronic stress persisted beyond the period of the actual rat exposure. (2) Post-training self-administration of sugar as reinforcer improved spatial performance in naïve mice, whereas (3) in stressed mice sugar partially “normalized” the impaired performance to the level of controls without sugar. Chronic stress (4) increased behavioral inhibition in response to novelty; (5) induced dynamic changes in the pattern of circadian corticosterone secretion during the first week after rat stress and (6) increased the intake of sucrose and water. (7) Chronic stress and sugar consumed during spatial training facilitated the memory for the location of the sucrose bottle weeks later. Concluding, our chronic stress paradigm induces the expression of anhedonia in mice, at different levels of behavior. The behavioral inhibition appears to be long lasting in stressed mice. Interestingly, sugar consumed in close context with spatial learning partially rescued the stress-induced emotional and cognitive impairments. This suggests that reward can ameliorate part of the negative consequences of chronic stress on memory.

Swim posture of mice does not affect performance in the water maze

We quantified swim postures of mice in relation to their cognitive performance. After training in a water maze, young (5-6 months) and aged (14-16 months) female apolipoprotein E-knockout (apoE0/0) mice and wild type controls were video taped while swimming. Subsequently, angles of body points with the water surface were calculated. Mice with a more horizontal swim posture (young and aged apoE0/0, aged wild type mice) also showed an increased body weight. However, swim posture was not related to cognitive performance.