Mechthild Voits

Perinatal elevation of hypothalamic insulin, acquired malformation of hypothalamic galaninergic neurons, and syndrome X-like alterations in adulthood of neonatally overfed rats

Overnutrition during critical developmental periods is suggested to be a risk factor for obesity and associated metabolic disorders in later life. Underlying mechanisms are unknown. Neuropeptides are essentially involved in the central nervous regulation of body weight. For instance, hypothalamic galanin (GAL) is a stimulator of food intake and body weight gain. To investigate long-term consequences of early postnatal overfeeding, the normal litter size of Wistar rats (n=10; controls) was reduced from day 3 to day 21 of life to only 3 pups per mother (small litters, SL; overnutrition). Throughout life, SL rats displayed hyperphagia (p<0.01), overweight (p<0.0001), hyperinsulinemia (p<0.01), impaired glucose tolerance (p<0.001), elevated triglycerides (p<0.001), and an increased systolic blood pressure (p<0.05). In adulthood, an increase of GAL-neurons in the arcuate hypothalamic nucleus (ARC) was found (p<0.001), positively correlated to body weight (p<0.001). A second experiment revealed hyperinsulinemia (p<0.001) and increased hypothalamic insulin levels (p<0.05) in SL rats during early postnatal life. Already on day 21 of life, i.e., at the end of the critical hypothalamic differentiation period, in SL rats the number of GAL-neurons was increased in the ARC (p<0.001), showing a positive correlation to body weight and insulin (p<0.05). In conclusion, neonatally acquired persisting malformation of hypothalamic galaninergic neurons, induced by early overfeeding and hyperinsulinism, might promote the development of overweight and syndrome X-like alterations during life.

Major biological actions of CCK – a critical evaluation of research findings

Abstract Cholecystokinin (CCK) is one of the first discovered gastrointestinal hormones and one of the most abundant neuropeptides in the brain. Two types of CCK receptors have been identified: (1) CCK-A receptors are mainly located in the periphery, but are also found in some areas of the CNS; and (2) CCK-B receptors are widely distributed in the brain. Major biological actions of CCK are the reduction of food intake and the induction of anxiety-related behavior. Inhibition of feeding is mainly mediated by the A-type receptors, whereas anxiety-like behavior is induced by stimulating B-type receptors. This paper presents new findings on the effects of the biologically active CCK agonists, CCK-8S, CCK-4, and A71378. The results reviewed suggest that the hypophagic effects of CCK are strongly dependent on the experimental design, sex, and age of the rats. For example, food intake measured during the night or after food deprivation is reduced by CCK-8S in young adult and aged rats, whereas, under fixed feeding conditions, CCK-8S does not inhibit food intake in young adult rats. The sensitivity to the hypophagic CCK effect increases with age in male and female rats; however, female rats are less sensitive to the CCK action. Further, using a nongenetic and non-stressful model of obesity due to unspecific postnatal overfeeding, the satiating effect of moderate CCK-8S doses is weaker in obese than in normal rats. Again, the hypophagic effect is more pronounced in male than in female obese and normal rats. Considering that aversive reactions in rats are markedly influenced by strain and breeding-line variations, research results in this area are critically reviewed. It is shown that anxiety-like symptoms can only be induced by a selectively acting CCK-B agonist, whereas mixed CCK-A and -B agonists and selective CCK-A agonists fail to change behavior in anxiety tests. CCK-4 induces stable and reproducible anxiogenic-like behavior only in certain rat strains. Moreover, CCK-4 effects can be demonstrated in the conflict test, in the ultrasonic vocalization test in rat pups, on the elevated plus maze, and in the black and white box, but not in the social interaction test. CCK has also been reported to modulate memory processes. On the one hand, CCK-8S and CCK-4 enhanced habituation to the novelty of a hole board. On the other hand, repeated administration of CCK-8S did not improve maze performance in aged rats. The literature on the behavioral pharmacology of CCK is rife with inconsistency and contradiction. The major biological actions of CCK depend on the receptor selectivity of the CCK fragments used and on organismic and procedural variables. All these variables potentially influence behavioral responses in rats. Therefore, in CCK research more attention should be paid to the importance of these methodological factors.

Fischer 344 and wistar rats differ in anxiety and habituation but not in water maze performance.

The fact that various neuropharmacological substances have anxiolytic as well as amnesic effects suggests that neuronal mechanisms of anxiety and learning/memory closely interact. Hence, we hypothesized that differences in anxiety-related behavior could be accompanied with differences in cognition or habituation. Two rat strains with different levels of anxiety, more anxious Fischer 344 rats by Charles River (FC) and less anxious Wistar rats by Winkelmann (WW), were tested in the Morris water maze task and an open field test for habituation learning. Additionally, we investigated the effect of different light intensities on the performance in the Morris water maze and the elevated plus maze. The results of the water maze task indicate that differences in anxiety-related behavior do not go along with differences in this performance of learning/memory. Moreover, the test was not affected by different light intensities. In contrast, illumination did affect performance in the elevated plus maze test, wherein dim light provoked an anxiolytic effect in both rat strains. The findings that neither different baseline levels of anxiety nor fear modulating light conditions were accompanied by changes in the performance of rats in the Morris water maze led us to the suggestion that there is no connection between anxiety and learning/memory in this task. Contrarily, anxiety might be associated with habituation learning in the open field test, shown by the superior habituation of the anxious FC rats in comparison to the less anxious WW rats. In sum, these results indicate that anxiety and learning/memory seem to be independently regulated behaviors, whereas habituation might be more closely correlated with anxiety. Nevertheless, a general statement about the relation between emotionality and learning/memory mechanisms would be premature and the link between behaviors remains to be clarified.

Evidence for mnemotropic action of cholecystokinin fragments Boc-CCK-4 and CCK-8S

Memory-modulating and reinforcing effects of the cholecystokinin (CCK) fragments, CCK-8S and Boc-CCK-4, after systemic application in rats were investigated. Habituation to the novelty of environmental stimuli was used to test for mnemonic effects using two different tasks (rearing behavior in an open field; head-dips in a hole-board). Immediate posttrial administration of CCK-8S and Boc-CCK-4 resulted in a reduction of rearing and head-dip behavior during testing, indicative of enhanced habituation and, thus, facilitation of memory. In contrast, administration of CCK-8S and Boc-CCK-4 with a delay of 2.5 or 5 h after training or pretrial injection of CCK-8S did not enhance habituation. No evidence for reinforcing or aversive properties of CCK-8S and Boc-CCK-4 was observed in a conditioned place preference task. In summary, the results indicate memory-enhancing effects of peripherally, posttrial-administered CCK-8S and Boc-CCK-4.

Effects of cholecystokinin octapeptide (CCK-8) on food intake in adult and aged rats under different feeding conditions

The effects of CCK on food intake were investigated under fixed feeding conditions in comparison to a test meal taken after 16 h of food deprivation. The experiments were performed on young adult rats (8 weeks old) as well on aged rats (23 months old). Intraperitoneal CCK-8 (8 and 40 micrograms/kg) significantly reduced the size of a test meal following 16-h food deprivation. This effect was independent of the age of the rats. However, under fixed feeding conditions neither of the doses used in this study reduced food intake in the young adult rats, whereas the highest dose of 40 micrograms/kg did so in the aged rats. These results suggest that the hypophagic effect of exogenous CCK-8 depends on experimental conditions, food intake being reduced after a period of food deprivation but not under a fixed feeding regimen in adult animals. Furthermore, the data suggest that age is a factor contributing to the complex behavioral actions of CCK, because only old animals were more susceptible to an anorectic action of CCK under the fixed feeding schedule. An explanation may lie in an interaction of other known behavioral effects of CCK (e.g., anxiogenic, mnemonic action) with its effects under the different feeding schedules.

Lesion of the Median Raphe Nucleus: A Combined Behavioral and Microdialysis Study in Rats

The purpose of the present study was to investigate the behavioral consequences and the neurochemical correlates of a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the median raphe nucleus (MRN) in rats. Anxiety-related behavior was assessed in the elevated plus maze test on days 5, 14, and 21 after lesioning. In general, behavior of MRN-lesioned rats was unchanged when compared with sham-lesioned or untreated controls. Neurochemically, microinjection of 5,7-DHT into the MRN resulted in 87.5% depletion of hippocampal 5-HT content. Using the in vivo microdialysis technique, the exposure of 5,7-DHT-lesioned rats to the elevated plus-maze failed to increase extracellular 5-HT release (94%) in the hippocampus, as shown in sham-lesioned (150%) or untreated controls (194%). Moreover, application of fenfluramine (10 mg/kg, i.p.) evoked a 10-fold increase in hippocampal extracellular 5-HT levels in sham-lesioned animals, whereas in 5,7-DHT lesioned rats 5-HT was only slightly increased. The results demonstrate, that a marked reduction of 5-HT release from the MRN is not necessarily accompanied by anxiolytic-like behavior.

Obesity induced by unspecific early postnatal overfeeding in male and female rats: hypophagic effect of CCK-8S

The response to cholecystokinin (CCK) as a satiety peptide in obesity or anorexia has been tested mainly in extreme models of food intake control. In the present study, the effect of CCK-8S on food intake was investigated in a nongenetic and less-stressful model of obesity due to unspecific early postnatal overfeeding in male and female rats. Reducing the normal litter size of ten to three newborn rats on day 3 of life led to an enhanced food intake resulting in an increased body weight until adulthood. Freely fed male and female, normal and obese rats were given 10 μg/kg CCK-8S i.p. on day 41 and 40 μg/kg CCK-8S on day 91 of life and food intake was measured for 24 h. Compared with treatment with saline (i.p.) 1 day before the test, the lower dose of 10 μg/kg CCK-8S reduced food intake for 2 h in normal, but not in obese rats. Conversely, the higher dose of 40 μg/kg CCK-8S reduced food intake in both normal and obese rats for 2 h, but this effect was more evident in the obese rats. Moreover, the satiating effect of CCK-8S was more pronounced and longer lasting in male than in female rats. In summary, the data suggest that the response to CCK-8S differs in normal and obese rats and depends on sex.

Application of ‘nose-poke habituation’ validation with post-trial diazepam- and cholecystokinin-induced hypo- and hypermnesia

The present study describes the use of nose-poke habituation as a memory task and demonstrates that it is sensitive to hypo- and hypermnestic pharmacological treatments administered post-trial. Habituation of nose-poke behavior of rats was defined as a reduction in number of nose-pokes compared to baseline. It was measured using a board with 16 holes, to which animals were exposed on 2 consecutive days (baseline and test) for 10 min, respectively. After the first exposure, rats were injected intraperitoneally (i.p.) immediately or with a delay of 2.5 h with doses of diazepam (0.9-4.5 mg/kg) known to be hypomnestic, or cholecystokinin (CCK-8S; 0.2-25 micrograms/kg), which was reported to have memory facilitating effects. An enhancement of habituation in comparison with vehicle controls was interpreted in terms of a hypermnestic effect of the treatment. Conversely, hypomnestic action of the drug treatment was inferred from a reduced habituation. The results show that when diazepam was injected immediately post-trial, the normal reduction in number of nose-pokes during test was prevented, indicative of a failure to habituate presumably due to an amnesia for the baseline/training trial. In contrast, enhanced habituation (facilitation of memory) was induced when CCK-8S was injected immediately post-trial, as reflected by a decrease in number of nose-pokes during test compared to control animals. The effects were not due to enduring proactive effects of the compounds on performance during test, since post-trial injections of diazepam or CCK-8S with a delay of 2.5 h did not have the effects that immediate post-trial injection had.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural and microdialysis study after neurotoxic lesion of the dorsal raphe nucleus in rats.

The study investigated the effects of a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsal raphe nucleus (DRN) on anxiety-related behaviour and neurochemical correlates in rats. Behaviour was assessed in the elevated plus maze test (X-maze). Lesion of the DRN reduced markedly 5-HT levels in projection areas by at least 60%. Destruction of the serotonergic neurons in the DRN changed neither anxiety-related behaviour on the elevated plus maze, nor aversion-induced 5-HT release in the brain. Exposure of the lesioned rats to the elevated plus maze increased extracellular 5-HT (148%) in the ventral hippocampus similar as in sham-lesioned (162%) and non-lesioned (160%) controls. The results demonstrate that lesioning of 5-HT neurons in the DRN does not abolish totally the control of anxiety-related behaviour.

Does increased endogenous CCK interact with serotonin to reduce food intake in rats

The present study was aimed to test the hypothesis that increased endogenous CCK may interact with the anorectic serotonergic agent dl-fenfluramine to reduce food intake in rats. Previous studies, using selective CCK receptor antagonists, could demonstrate CCK-dependent 5-HT-induced anorexia. In the present approach, we used protease inhibitors to increase levels of endogenous CCK instead of blocking CCK receptors by antagonists. The protease inhibitors we used were soybean trypsin inhibitor (STI) and camostate. We hypothesized that combining the anorectic serotonergic drug dl-fenfluramine with either STI or camostate should result in an enhanced hypophagic effect when compared to single drug treatment. All feeding experiments were performed in non-deprived rats during night time feeding. Given alone, STI (500 mg/kg, po), camostate (200 mg/kg po) and also fenfluramine (1-9 mg/kg ip) reduced significantly food intake, with a more pronounced effect following fenfluramine. However, the experiments do not provide evidence for any additive or synergistic action between camostate or STI and the anorectic serotonergic drug dl-fenfluramine on food intake.