Central metabolite changes and activation of microglia after peripheral interleukin-2 challenge.

Interleukin (IL)-2 regulates the immune response through the proliferation of activated T-cells and also exerts effects on the central nervous system (CNS). Alongside having marked neurobehavioral effects, IL-2 has been suggested to impact on various psychiatric disorders. The immune-CNS communication of IL-2 remains unclear, although, it is suggested that microglia are the source and target of IL-2. Here, we analyzed changes in brain metabolites following a peripheral IL-2 challenge and examined the contribution of microglia in mediating these effects. Rats were assessed by magnetic resonance spectroscopy (MRS) in a 9.4 T scanner for baseline metabolite levels in the prefrontal cortex (PFC) and the hippocampus. After 7 days animals were scanned again following a single injection of IL-2 (2.5 µg/kg) and then tested on the elevated plus-maze for the correlation of IL-2-induced brain metabolites and measures of anxiety. In another experiment CD25(+) microglia cells were determined. A separate group of rats was injected either with IL-2 or vehicle, and afterward the PFC and hippocampus were dissected and fluorescence activated cell sorting (FACS) analysis was performed. The MRS scans in the intra-individual study design showed a significant increase in myo-inositol in the analyzed regions. A significant correlation of anxiety-like measures and myo-inositol, a marker for microglia activity, was found in the hippocampus. The FACS analysis showed a significant increase in CD25(+) microglia in the hippocampus compared to controls. The results support the role of microglia as a mediator in the immune-CNS communication and the effects of peripheral IL-2.

Behavioral and IL-2 responses to diosgenin in ovariectomized rats.

Neuroimmune system is involved in communication between the endocrine and nervous systems, which may take part in the effects of dioscorea, reversing changes of anxiety-like behavior and interleukin (IL)-2 levels in the brains of ovariectomized (OVX) rats. This study was aimed at evaluating administration of diosgenin, an ingredient of dioscorea, on neuroimmune and behavioral functions in OVX animals. One month after ovariectomy, female Wistar rats were fed daily with diosgenin (0, 10, 50 or 100 mg/kg/day) and the elevated plus-maze and learned helplessness tests were used to measure anxiety-like and depressive behaviors after 23 and 24 days of diosgenin treatment, respectively. In the learned helplessness test, the rats needed to cross from one compartment of the shuttle box to the opposite compartment to avoid or escape the shock. If the rat failed to escape the shock in 10 sec, a "failure" was recorded. Two days after the behavioral tests, the brain was removed to measure levels of IL-2 which was used as an indicator of neuroimmune function. Anxiety-like behavior in the OVX rats was not affected by diosgenin treatment. However, avoidance behavior in the learned helplessness test in the OVX rats with high anxiety (HA) levels was improved by treatment with diosgenin at the dosage of 10mg/kg/day. Interestingly, the number of failures in the same test was increased when the dosage of diosgenin was increased to 50 mg/kg/day, and this was accompanied by an increase in IL-2 levels in the pituitary gland. In addition, treatment with 100 mg/kg/day of diosgenin resulted in decreased IL-2 levels in the amygdala and prefrontal cortex of the OVX rats with low anxiety levels, and in increased IL-2 levels in the amygdala of OVX HA rats. These results show that chronic diosgenin treatment influences IL-2 levels in the brain of OVX rats and affects depressive behavior in OVX HA rats, but not OVX low anxiety rats.

Animal models of human psychopathology based on individual differences in novelty-seeking and anxiety.

The role of individual factors in behavioural neuroscience is an important, but still neglected area of research. The present review aims to give, first, an outline of the most elaborated theory on animal behaviour, and second, an overview of systematic approaches of historic and present animal models of human psychopathology based on individual differences. This overview will be focused on animal models of unselected subjects (i.e. natural variance of a specific behaviour within a given population) and selected breeding for a specific behaviour. Accordingly, an outline of the personality model from Gray and McNaughton of individual behaviour in animals is given first. Then, a comprehensive overview of past and current animal models in novelty-seeking (i.e. psychomotor activation and exploration behaviour) based on systematic individual differences and its relationship to addiction is presented. Third, this will be followed by a comprehensive overview of individual differences in previous and present animal models for anxiety. Finally, critical aspects of such approaches in animal research are discussed, and suggestions are given where to go from here.

Striatal microinjections of interleukin-2 and rat behaviour in the elevated plus-maze.

We showed that the relationship between cytokine mRNA in the rat brain and elevated plus-maze behaviour is site- (striatum, prefrontal cortex), and cytokine-specific (interleukin-2). Here, we investigated whether a striatal microinjection of interleukin-2 (1, 10, 25 ng) affects elevated plus-maze behaviour. Analyses showed no acute effects of IL-2 on open arm time, whereas dose-dependent differences in rearing activity, and open arm entries became apparent between IL-2 doses. Twenty-four hours later, a previous dose of 25 ng IL-2 showed a trend for more open arm time compared to vehicle. These behavioural changes are discussed in relation to anxiety-relevant and exploratory behaviour, and possible neurochemical mechanisms.

Cytokine mRNA levels in brain and peripheral tissues of the rat: relationships with plus-maze behavior.

There is evidence that interleukin (IL)-2 may be related to anxiety as measured in the elevated plus-maze. Recently, we showed that normal adult male Wistar rats can differ systematically in this test of avoidance behavior, that is, time spent on the open arms of the elevated plus-maze. Rats with low open arm time had higher striatal levels of IL-2 mRNA than those with high open arm time, but did not differ significantly in expression of other striatal cytokine mRNA. Here, we investigated whether these expression effects are anatomically specific to the striatum. Therefore, we asked in this double-blind study whether elevated plus-maze behavior may also be related to endogenous levels of cytokine mRNA in other brain regions, which play a role for anxiety, namely the amygdala, hippocampus, and the prefrontal cortex. Additionally, and as peripheral controls, immuno-neuro-endocrine relevant tissues (adrenal glands, spleen) were analyzed. Based on open arm time in the elevated plus-maze, male Wistar rats were divided into sub-groups with either low or high open arm time behavior. Then, IL-1beta, IL-2, IL-6, and tumor necrosis factor (TNF)-alpha cDNA levels were measured post-mortem using semi-quantitative, competitive, reverse transcription polymerase chain reaction. First, we found that cytokine expressions differed considerably between and within these central and peripheral tissues. Secondly, rats with high compared to low open arm time behavior showed higher IL-2 mRNA levels in the prefrontal cortex, which is an inverse pattern to what we recently found in the striatum. These results provide new evidence indicating that cytokine mRNA in the brain can be related to elevated plus-maze behavior and that this relationship is site (prefrontal cortex, striatum)- and cytokine mRNA-specific (IL-2).

Repeated nicotine treatment in rats with high versus low rearing activity: analyses of behavioural sensitisation and place preference.

RATIONALE: Repeated treatment with the cholinergic agonist nicotine can sensitise rats to its psychomotor stimulant effects, which is largely due to changes within the mesolimbic and mesostriatal dopamine system. Since this brain system also plays a critical role in motivational processes, changes of motivational functions may also be expected with repeated nicotine experiences. OBJECTIVE: Our previous work has shown that normal male Wistar rats can differ systematically with respect to rearing activity in a novel open field: animals with high rearing activity (HRA) differed from those with low rearing activity (LRA) with respect to dopaminergic and cholinergic brain activity. In this study, we asked whether HRA and LRA rats might respond differentially to repeated nicotine treatment, which we tested in terms of behavioural sensitisation and place preference. METHODS: Nicotine hydrogen tartrate (0.4 mg/kg) or saline was administered on eight alternate days (drug treatment). After each injection, the rats had access to one specific quadrant of a circular unbiased place preference apparatus. Sensitisation to nicotine was assessed by measuring locomotion and rearing during drug treatment. On the days after each drug treatment, rats had free access to the entire apparatus without prior drug treatment. Here, we tested for preference for the previously drug-paired quadrant. One week after this procedure, all animals were tested again for sensitisation and place preference after injection of saline or nicotine. RESULTS: Overall, sensitisation occurred earlier during locomotor than rearing activity. Both, HRA and LRA rats treated with nicotine showed sensitisation, but with different profiles. Rearing sensitised earlier in HRA than LRA rats, and a sensitised locomotor response to nicotine was observed only in HRA rats when compared with baseline. When re-tested again 1 week later, expression of sensitisation to nicotine was detected in rearing and locomotor activity in both HRA and in LRA rats. In the place preference tests, nicotine-treated and saline-treated rats spent more time in the treatment quadrant, but nicotine did not lead to place preference compared to saline. Furthermore, there was no substantial evidence that nicotine might lead to place preference in only HRA or LRA rats. However, we obtained other evidence that HRA versus LRA rats responded differently to the procedure of place preference testing. CONCLUSIONS: These data supplement previous findings that different levels of psychomotor activity can affect the reactivity to psychostimulant drugs and add new evidence with respect to nicotine.

Acute and long-term consequences of single MDMA administration in relation to individual anxiety levels in the rat.

Our previous work has shown that normal male Wistar rats can differ systematically in their behavioral response to the elevated plus-maze (EPM), where animals with high (HA) or low anxiety (LA) levels can be identified based on the percentage of time spent in the open arms. These animals also differ in other behavioral tests (e.g. active avoidance), and in their serotonin levels in the ventral striatum. Here, we tested whether such HA and LA rats might respond differently to the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). This drug can affect psychomotor activation and anxiety; effects which are probably due to its pronounced serotonergic and dopaminergic impacts in the rat brain. Based on a routine screening procedure in the plus-maze, male Wistar rats were divided into HA and LA sub-groups, in which rectal temperature was measured. Thirty minutes after the i.p. injection of MDMA (7.5 or 15 mg/kg) or vehicle, they were again tested in the plus-maze. During the next 3 weeks, the animals underwent further behavioral tests (plus-maze, open field, active avoidance, forced swimming) to test for possible long-term consequences of MDMA. Rectal temperature was found to be higher in LA than HA rats and was especially increased with the higher dose of MDMA (15 mg/kg). In the acute plus-maze test, the lower dose of MDMA led to an anxiogenic-like profile, whereas the higher dose led to an anxiolytic-like profile, both in HA and LA rats. Possible long-term consequences of MDMA were only tested with 7.5 mg/kg MDMA, since the 15 mg/kg dose led to a high level of lethality. The analysis of open field, plus-maze (performed after 9-12 days), and forced swimming behavior (performed after 20-21 days) did not provide indications for lasting effects of MDMA. In contrast, active avoidance learning was impaired in LA- but not HA-rats treated with MDMA. A single injection of MDMA does not only have acute effects on anxiety and psychomotor activation, but can also have some prolonged or delayed task-dependent behavioral consequences. The detection of such sequels can require that individual differences are taken into account and here, determining anxiety levels in the EPM seems to serve as a useful approach.

Relationship between striatal levels of interleukin-2 mRNA and plus-maze behaviour in the rat.

Our previous experiments have shown that adult male Wistar rats can differ systematically in elevated plus-maze (EPM) behaviour, which was related to the neurotransmitter serotonin in the ventral striatum. The EPM serves as a model of anxiety-like behaviour, and there is evidence that interleukin (IL)-2 in the brain may be related to anxiety-like behaviour, and that IL-2 interacts with the striatal serotonergic system. We asked whether EPM behaviour may also be related to constitutive levels of cytokines in the striatum. Based on open arm time in the EPM, male Wistar rats were divided into sub-groups with either low or high anxiety-like behaviour. Then, IL-1beta, IL-2, IL-6, and tumour necrosis factor (TNF)-alpha cDNA levels were measured post mortem in striatal tissues using semi-quantitative, competitive, reverse transcription polymerase chain reaction. Rats with high anxiety-like behaviour in the EPM showed significantly higher levels of IL-2 mRNA compared to those with low anxiety-like behaviour, but did not differ significantly in expression of IL-1beta, IL-6, and TNF-alpha mRNA. These results provide new evidence indicating that specific cytokine patterns in the striatum may be associated with EPM behaviour in adult male Wistar rats.

Object preference and nicotine consumption in rats with high vs. low rearing activity in a novel open field.

Our previous work has shown that normal male wistar rats can differ systematically with respect to rearing activity in a novel open field: animals with high rearing activity (HRA rats) differed from those with low rearing activity (LRA rats) in dopaminergic and cholinergic brain activity, as well as in their behavioral responsiveness to a cholinergic antagonist, but not in measures of anxiety in the elevated plus-maze. Here, we tested (a) whether HRA vs. LRA reflects responsiveness to novelty, (b) whether such rats voluntarily consume different amounts of the cholinergic agonist nicotine and (c) whether these measures are related to those of anxiety in the plus-maze. Using a novel object test, we found that HRA showed a trend for more object exploration than LRA rats when confronted with two identical novel objects in a familiar open field. When subsequently confronted with a familiar vs. a new object, HRA rats showed substantially more exploration of the new but not of the familiar object than LRA rats. In a subsequent test, HRA vs. LRA rats did not differ in voluntary or forced consumption of oral nicotine, or water. In contrast to rearing activity in a novel open field, measures of anxiety in the plus-maze were neither related to behavior in the novel object test nor to voluntary oral consumption of nicotine, or water. Among others, these data are discussed with respect to dopaminergic and cholinergic forebrain mechanisms, which have previously been found to differ between HRA and LRA rats. Since forebrain dopamine and acetylcholine functions are critical for novelty processing, we suggest that they are also important for the differential behavioral patterns of HRA and LRA rats in the open field, and in the novel object test.

Time-dependent effects of striatal interleukin-2 on open field behaviour in rats.

There is evidence that immune messengers like cytokines can modulate emotional and motivated behaviours and are involved in psychiatric conditions like anxiety, and depression. Previously, we showed that cytokine gene expression (interleukin (IL)-2 mRNA) in specific brain tissues (striatum, prefrontal cortex) correlated with anxiety-like behaviour (open arm time) in an elevated plus-maze in rats. In a subsequent experiment, a single striatal IL-2 injection showed behavioural trends with the lower dose (1 ng) acting in a behavioural suppressive way, whereas the highest dosage (25 ng) led to activation and anxiolytic-like behaviour. Here, to support and extend our previous findings, we tested Wistar outbred rats after a single unilateral (balanced brain sites) IL-2 injection into the ventral/dorsal striatum followed by an open field test acutely and 24 h later. Analyses for horizontal locomotion showed no differences between groups. However, rats with IL-2-treatment (0.1 ng) showed a dose-dependent avoidance effect (i.e. reduced centre time) compared to the 1 ng group and vehicle controls 24 h later. In addition, suppression of free rearing activity was shown for both IL-2 doses (0.1; 1 ng) compared to saline in the acute test, and partly 24 h later. Thus, in experiment 2, we tested for proactive drug mechanisms to test for delayed effects of IL-2 as observed in experiment 1. In a new sample, rats were returned to their home cages after a striatal IL-2 injection (0.1; 0.01; 0 ng), and tested 24 h and 48 h after the injection in an open field. Neither for the first (24 h) nor for the second exposure (48 h later) did the analyses show any significant behavioural effects. We therefore suggest that emotional-related behaviour can be modulated by striatal IL-2 for at least 24 h. However, such IL-2 effects can only be observed if a mild stressful environmental challenge (i.e., forced open field exposure) is followed immediately after injection. In conclusion, proactive drug effects may be excluded for striatal IL-2 effects on open field behaviour.

Cue-induced alcohol-seeking behaviour is reduced by disrupting the reconsolidation of alcohol-related memories.

RATIONALE: In humans, the retrieval of memories associated with an alcohol-related experience frequently evokes alcohol-seeking behaviour. The reconsolidation hypothesis states that a consolidated memory could again become labile and susceptible to disruption after memory retrieval. OBJECTIVES: The aim of our study was to examine whether retrieval of alcohol-related memories undergoes a reconsolidation process. METHODS: For this purpose, male Wistar rats were trained to self-administer ethanol in the presence of specific conditioned stimuli. Thereafter, animals were left undisturbed in their home cages for the following 21 days. Memory retrieval was performed in a single 5-min exposure to all alcohol-associated stimuli. The protein synthesis inhibitor anisomycin, the non-competitive N-methyl-D: -aspartate (NMDA) receptor antagonist MK-801 and acamprosate, a clinically used drug known to reduce a hyper-glutamatergic state, were given immediately after retrieval of alcohol-related memories. The impact of drug treatment on cue-induced alcohol-seeking behaviour was measured on the following day and 7 days later. RESULTS: Administration of both anisomycin and MK-801 reduced cue-induced alcohol-seeking behaviour, showing that memory reconsolidation was disrupted by these compounds. However, acamprosate had no effect on the reconsolidation process, suggesting that this process is not dependent on a hyper-glutamatergic state but is more related to protein synthesis and NMDA receptor activity. CONCLUSIONS: Pharmacological disruption of reconsolidation of alcohol-associated memories can be achieved by the use of NMDA antagonists and protein synthesis inhibitors and may thus provide a potential new therapeutic strategy for the prevention of relapse in alcohol addiction.

A phenotype-driven ENU mutagenesis screen for the identification of dominant mutations involved in alcohol consumption.

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an "alcohol phenotype," that is, high or low ethanol intake. They were then selected for breeding and tested in a "confirmation cross" (G2-G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.

Effects of escapable and inescapable stressors on behavior and interleukin-2 in the brain.

This study aimed to clarify the effects of inescapable and escapable stressors on behavior and interleukin-2 (IL-2) levels in the brain. Inescapable trials, consisting of pairings of conditioned and unconditioned stimuli, were used to induce fear-conditioned stress, whereas trials of escapable pairings of conditioned and unconditioned stimuli in an active avoidance test were used as acute and conditioned stressors. IL-2 levels in the brain were analyzed by enzyme-linked immunoadsorbent assays. Inescapable and escapable stressors had different effects on behavior in the modified active avoidance test and on IL-2 levels in brain areas that are known to be involved in emotional processes. These data provide insight into the pathophysiological role of IL-2 in stress-related disorders.

Flares in patients with systemic lupus erythematosus are associated with daily psychological stress.

BACKGROUND: The aetiology of systemic lupus erythematosus (SLE) remains unclear. Clinical observations and a small number of studies performed so far suggest an association between psychological stress and self-reported symptoms of SLE patients. This longitudinal study was designed to investigate whether daily psychological stress is associated with flares in SLE patients, measured by clinical and laboratory parameters. METHODS: Female SLE patients (n = 41) were followed over a period of six months. Daily stress was monitored by a hand-held PC diary programmed with 44 items based on standardized measures and clinical experience. Once every four weeks patients visited the outpatient clinic for medical evaluation. Disease activity was evaluated using the European Consensus Lupus Activity Measurement (ECLAM), laboratory parameters, and intake of steroids. RESULTS: Classification and regression tree (CART) patient-wise analyses revealed that SLE patients with vs. without flares using complement and ECLAM as activity measures show greater negative self-ratings in mood, and social duties (p < 0.01). In addition, mixed model analysis of variance showed that daily hassles with social relationships were significantly associated with flares in SLE measured by an increase in steroid medication >5mg/d (p < 0.01). CONCLUSIONS: These results suggest that psychological stress is associated with flares in SLE. Particularly daily stress with social relationships and social duties may be factors to be related to the course of disease activity in SLE.