Early postnatal exposure to a cafeteria diet interferes with recency and spatial memory, but not open field habituation in adolescent rats.

The cafeteria diet (CD), an experimental diet that mimics the obesogenic Western diet, can impair memory in adult rats. However, the suckling period is also particularly susceptible to diet-induced behavioural modification. Here, following exposure to CD feeding during lactation, 24- to 26-day-old offspring were tested to determine maternal dietary effects on either open field habituation, object location (OL) learning or on recency learning. Whereas no impact on habituation learning could be demonstrated, both OL and recency memory were impaired. In controls (C), OL memory was shown both after a 5 min (p < .05) or 60 min (p < .001) inter-trial interval (ITI). After the 60 min ITI, the difference between C and CD was significant (p < .05). Learning did not occur in the CD group at any time point and was not observed after the 24hr ITI in in either group. Whereas control rats demonstrated intact recency memory (p < .00001), no learning occurred in the CD group. Both groups differed significantly in their exploration ratios (p < .01). This study suggests a detrimental effect of exposure to an unhealthy Western diet during lactation, on cognitive functions in adolescent rats. These results could have implications for human cognition in the context of obesity epidemic.

Impact of cafeteria feeding during lactation in the rat on novel object discrimination in the offspring.

There is increasing evidence that hyperenergetic diets have an impact on memory in rodents. However, it is largely unknown how diets, such as a cafeteria diet (CD), that mimic a Western-type diet act on learning and memory, in particular when fed during early stages of development. Here, we fed lactating dams a CD and exposed both male and female offspring to a novel object discrimination (NOD) task, a two-trial test of recognition memory in which rats exposed to two identical objects during a training/familiarisation trial can discriminate a novel from a familiar object during the subsequent choice trial. The choice trial was performed following inter-trial interval (ITI) delays of up to 4 h. Maternal diet did not have an impact on exploration of the objects by either sex during the familiarisation trial. Control males discriminated the novel from the familiar object, indicating intact memory with an ITI of 1 h, but not 2 or 4 h. The CD delayed this natural forgetting in male rats such that discrimination was also evident after a 2 h ITI. In contrast, control females exhibited discrimination following both 1 and 2 h ITI, but the CD impaired performance. In summary, the present study shows that maternal exposure to the CD programmes NOD in the adult. In better-performing females, dietary programming interferes with NOD, whereas NOD was improved in males after lactational CD feeding.

Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

Impaired spatial memory and altered dendritic spine morphology in angiotensin II type 2 receptor-deficient mice.

Mental retardation is the most frequent cause of serious handicap in children and young adults. Mutations in the human angiotensin II type 2 receptor (AT2) have been implicated in X-linked forms of mental retardation. We here demonstrate that mice lacking the AT2 receptor gene are significantly impaired in their performance in a spatial memory task and in a one-way active avoidance task. As no difference was observed between the genotypes in fear conditioning, the detected deficit in spatial memory may not relate to fear. Notably, receptor knockout mice showed increased motility in an activity meter and elevated plus maze. Importantly, these mice are characterized by abnormal dendritic spine morphology and length, both features also found to be associated with some cases of mental retardation. These findings suggest a crucial role of AT2 in normal brain function and that dysfunction of the receptor has impact on brain development and ultrastructural morphology with distinct consequences on learning and memory.

Increasing the number of 5-HT(1A)-receptors in cortex and hippocampus does not induce mnemonic deficits in mice.

Even though the role of the serotonin1A (5-HT(1A))-receptor for cognitive processes is undisputed, the exact involvement of pre- and postsynaptic sites remains unexplained. Recently, we introduced a mouse line overexpressing the 5-HT(1A)-receptor in the hippocampus and cortex. In this study we investigated in comparison to wild-type mice their cognitive abilities using the Morris water-maze task and inhibitory avoidance test. Acute effects of pre- and posttraining administered 8-OH-DPAT (0.03-0.3 mg/kg i.p.) were examined in the inhibitory avoidance test. Additionally, habituation learning was studied in the hole-board test. Transgenic mice showed no overall learning deficit. Spatial learning and memory revealed in the Morris water-maze task was comparable to wild-type mice, and both genotypes habituated to the hole-board arena in a similar manner. Comparing the performance of both genotypes in the inhibitory avoidance test, cognitive functions of transgenic mice seemed to be slightly impaired. When 8-OH-DPAT was administered pretraining an amnesic effect was produced only in transgenic mice and only at the highest dose (0.3 mg/kg). Posttraining administered 0.3 mg/kg 8-OH-DPAT did not affect the performance of both genotypes. Overall, the cortical and hippocampal overexpression of the 5-HT(1A)-receptor had no major effect on cognitive functions in mice, suggesting that changes in the 5-HT(1A)-receptor density are not necessarily accompanied with alterations of learning and memory processes.

The mTPH2 C1473G single nucleotide polymorphism is not responsible for behavioural differences between mouse strains.

Tryptophan hydroxylase 2 (TPH2) is the rate limiting enzyme of serotonin synthesis in the brain. A recently described functional (C1473G) single nucleotide polymorphism in mouse TPH2 resulting in vitro in a strongly decreased enzymatic activity was suspected to be responsible for the observed differences in 5-HT levels and behaviour between mice strains. We bred two substrains of C57BL/6 mice carrying the two isoforms and could show that both exhibit equal TPH activity, brain 5-HT content and behaviour. These data indicate that the distinct behavioural characteristics of mouse strains are not due to differences in TPH2 activity, but to other variations in the genetic background.

Hypophagic effect of the angiotensin AT1 receptor antagonist irbesartan in rats.

Recent experimental and clinical studies report beneficial metabolic effects of antihypertensive drugs interfering with angiotensin. Antagonists at the angiotensin AT(1) receptor can reduce blood glucose and triglyceride levels. So far, there is little evidence, however, that angiotensin AT(1) receptor antagonists can also affect food intake. Particularly unknown is if drugs of this class can have acute effects on short term feeding. To address this issue, the angiotensin AT(1) receptor antagonist irbesartan was studied in a one-hour feeding paradigm in rats. In this study, irbesartan was investigated in comparison with fenfluramine, an established satiating drug, and the angiotensin converting enzyme (ACE) inhibitor captopril. We found a significant reduction of one-hour food intake following 100-200 mg/kg (i.p.) irbesartan. The ACE inhibitor captopril (25-100 mg/kg i.p.) remained without effect on food intake and fenfluramine showed the expected hypophagic action starting at 1 mg/kg (i.p.). The hypophagic effect of irbesartan could not be attributed to sedation or any gross effect on motor activity as determined both upon feeding and independent activity experiments. Fenfluramine (1 mg/kg) and irbesartan (100 mg/kg) did not reduce the latency to feed, but similarly reduced the eating rate at the beginning of the test meal. In conclusion, the present study demonstrates a hypophagic effect of the angiotensin AT(1) receptor antagonist irbesartan that cannot be attributed to sedation or antidipsic effects of the drug.

Exposure to a maternal cafeteria diet changes open-field behaviour in the developing offspring.

The early postnatal period is a sensitive period in rodents as behavioural systems are developing and maturing during this time. However, little is currently known about the behavioural effects of feeding a hyper-energetic cafeteria diet (CD) during the lactational period when offspring behaviour is tested during early adolescence. To this end, 23days old offspring from dams (Wistar) fed on CD during lactation were tested in either the open-field or the elevated plus-maze for exploration and anxiety-related behaviour. On postnatal day 9, maternal behaviour and non-maternal behaviour of the dam was assessed. It was hypothesized that lactational CD feeding would reduce anxiety in the offspring. CD-fed dams had a higher energy intake, due to an overconsumption of sugars and fats. When offspring from these dams were exposed to the open field after weaning, their locomotor activity was increased. They entered the more aversive inner zone of the open-field after a shorter latency, made more entries into and spent more time in the inner zone. Anxiety-related behaviour was not affected upon exposure to the elevated plus maze, suggesting anxiolysis in the open-field only. Increased maternal licking/grooming behaviour could possibly contribute to the anxiolytic phenotype as observed in the offspring from the CD group. In conclusion, we demonstrate that lactational overfeeding impacts on the development of behaviour in the early adolescent rat.

The impact of early postnatal environmental enrichment on maternal care and offspring behaviour following weaning.

The early postnatal period is a sensitive period in rodents as behavioural systems are developing and maturing during this time. However, relatively little information is available about the impact of environmental enrichment on offspring behaviour if enrichment is implemented only during this period. Here, environmental enrichment was provided from postnatal day 1 until weaning. On post-natal day 9, maternal behaviour and nonmaternal behaviour of the dam was observed. Nursing time in the enriched group was reduced but dams showed more non-maternal appetitive behaviours. Offspring were exposed to either the open field or the elevated plus maze (EPM) after weaning. In the open field, rats from the enriched group approached the more aversive inner zone of the open field later than control rats. Offspring from the enriched group made fewer entries into the inner zone and spent less time in this part of the arena. Enrichment had no impact on behaviour in the EPM. The present study provides evidence that postnatal enrichment can interfere with maternal behaviour in rats and can possibly lead to increased anxiety in the offspring. The findings suggest that enrichment procedures can have potentially unintended effects, interfering with the development of emotional behaviours in rats.

Food Deprivation, Body Weight Loss and Anxiety-Related Behavior in Rats.

In behavioral studies, food deprivation protocols are routinely used to initiate or maintain motivational states that are required in a particular test situation. However, there is limited evidence as to when food deprivation compromises animal welfare. This study investigated the effects of different lengths of food deprivation periods and restricted (fixed-time) feeding on body weight loss as well as anxiety-related and motivated behavior in 5-6 month old male and female Wistar rats. The observed body weight loss was not influenced by sex and ranged between 4% (16 h deprivation) to approximately 9% (fixed-time feeding). Despite significant body weight loss in all groups, the motivation to eat under the aversive test conditions of the modified open field test increased only after 48 h of food deprivation. Long-lasting effects on anxiety as measured in the elevated plus maze test 24 h after refeeding have not been observed, although fixed-time feeding could possibly lead to a lasting anxiogenic effect in female rats. Overall, female rats showed a more anxiolytic profile in both tests when compared to male rats. Despite these sex differences, results suggest that food deprivation is not always paralleled by an increased motivation to feed in a conflict situation. This is an important finding as it highlights the need for tailored pilot experiments to evaluate the impact of food deprivation protocols on animals in regard to the principles of the 3Rs introduced by Russell and Burch.

Brain angiotensin and anxiety-related behavior: the transgenic rat TGR(ASrAOGEN)680.

The transgenic rat TGR(ASrAOGEN)680, characterized by a transgene-producing antisense RNA against angiotensinogen in the brain, provides an opportunity to study the behavioral effects of angiotensin. While exposed to the elevated plus-maze (EPM) and the light/dark box, TGR(ASrAOGEN)680 rats showed more signs of anxiety compared to parental Sprague-Dawley (SD) rats. In the EPM, they made fewer entries into the open arms, spent less time there and more time on the closed arms. Head dips were reduced and U-turns were increased. In the light/dark box, the latency to the first re-entry into the light compartment was higher in TGR(ASrAOGEN)680. They displayed more SAP out from the dark and a reduced number of transitions between the two compartments. In the social interaction test, active social contacts were reduced, further suggesting an anxious phenotype. Although there was no transgenic effect on distance traveled in the open field, the more anxious TGR(ASrAOGEN)680 spent less time in the inner zone. Self-grooming was increased in TGR(ASrAOGEN)680 during exposure to the EPM and the open field, but was decreased in the social interaction test. In TGR(ASrAOGEN)680, tissue content of 5-HT and its metabolite 5-HIAA was lower in the hippocampus, frontal, and parietal cortex. HIAA and 5-HIAA/5-HT ratios were reduced in the hypothalamus, striatum, and septum. In the open field, the anxiogenic effect of the 5-HT2C/1B receptor agonist mCPP (0.5-1 mg/kg IP) was more pronounced in TGR(ASrAOGEN)680. The data suggest an anxious phenotype in rats with low brain angiotensinogen, possibly related to secondary dysfunctions of the brain serotonergic system.

Serotonin controlling feeding and satiety.

Serotonin has been implicated in the control of satiety for almost four decades. Historically, the insight that the appetite suppressant effect of fenfluramine is linked to serotonin has stimulated interest in and research into the role of this neurotransmitter in satiety. Various rodent models, including transgenic models, have been developed to identify the involved 5-HT receptor subtypes. This approach also required the availability of receptor ligands of different selectivity, and behavioural techniques had to be developed simultaneously which allow differentiating between unspecific pharmacological effects of these ligands and 'true' satiation and satiety. Currently, 5-HT1B, 5-HT2C and 5-HT6 receptors have been identified to mediate serotonergic satiety in different ways. The recently approved anti-obesity drug lorcaserin is a 5-HT2C receptor agonist. In brain, both hypothalamic (arcuate nucleus, paraventricular nucleus) and extrahypothalamic sites (parabrachial nucleus, nucleus of the solitary tract) have been identified to mediate the serotonergic control of satiety. Serotonin interacts within the hypothalamus with endogenous orexigenic (Neuropeptide Y/Agouti related protein) and anorectic (α-melanocyte stimulating hormone) peptides. In the nucleus of the solitary tract serotonin integrates peripheral satiety signals. Here, the 5-HT3, but possibly also the 5-HT2C receptor play a role. It has been found that 5-HT acts in concert with such peripheral signals as cholecystokinin and leptin. Despite the recent advances of our knowledge, many of the complex interactions between 5-HT and other satiety factors are not fully understood yet. Further progress in research will also advance the development of new serotonergic anti-obesity drugs.

Anxiolytic-like profile in Wistar, but not Sprague-Dawley rats in the social interaction test.

RATIONALE: The social interaction test is a valuable behavioural model for testing anxiolytic drugs in rodents, quantifying the level of social behaviour between pairs of rats. OBJECTIVE: The aim of the present study was to assess the appropriateness of the social interaction test for use with a Sprague-Dawley rat line, because of increasing use of this strain in targeted mutagenesis research. METHODS: Sprague-Dawley and Wistar rats received either diazepam or mCPP or were exposed to different environmental conditions (lighting, social isolation prior testing, habituation, testing-time). General anxiety-related parameters measured were: duration of active social contact, frequency of active social contact, latency to first contact. Different forms of active social contact were recorded: number of crawls, follows and sniffs. Secondly, aversion-induced hippocampal serotonin release and serotonin content in brain regions were measured. RESULTS: In Wistar rats habituation to the test substantially increased the time of social contact, an effect comparable with treatment with diazepam (1 mg/kg), whereas changes in the lighting level had less impact. Latency to the first contact increased under "anxiety-reducing" conditions, the frequency of contacts did not change consistently. Sprague-Dawley rats behaviour did not change under varying environmental conditions, and treatment with diazepam had only sedating effects at higher doses (5 mg/kg). Anxiogenic doses of mCPP caused reduced social interaction in both strains. Serotonin release and serotonin content were higher in the anxious Wistar rats. CONCLUSIONS: Different rat strains as well as differing test conditions have a major impact on the outcome of this animal test for anxiety.

The impact of cafeteria diet feeding on physiology and anxiety-related behaviour in male and female Sprague-Dawley rats of different ages.

There is emerging experimental evidence that hyper-energetic diets not only cause obesity but also impact on behaviour in rodents. A hyper-energetic comfort diet/cafeteria diet (CD) fed during early development programmes anxiety-related behaviour in adult age, but little is known how an obesogenic CD impacts on behaviour when fed at a later age. To this end we fed CD to Sprague-Dawley rats of both sexes at either 6 weeks or 12 months old, for a period of 6 weeks. Anxiety-related behaviour was assessed in the elevated plus maze (EPM) and the open field (OF). A glucose tolerance test was performed and metabolic indices, body weight and fat were measured. CD-fed young adult females, but not males, had a higher energy intake, due to an overconsumption of carbohydrates and fats. Only in adult CD-fed rats of both sexes did this overconsumption led to increased weight gain. Protein intake was reduced in all CD groups. Fat mass (subcutaneous, perirenal, gonadal) increased in most CD groups, whereas brown fat increased only in adults. Triacylglycerol, free fatty acid and total cholesterol concentrations increased predominantly in adult CD-fed rats. Glucose tolerance was only impaired in adult males. CD-fed adult males showed fewer entries into the aversive open arms and groomed more on the EPM, whereas adult females spent more time on these arms. In the OF, CD-fed females of both ages visited the inner zone more frequently and travelled a longer distance. The behavioural data suggests anxiolysis in CD-fed females and signs of increased anxiety in adult males. In conclusion, this study demonstrates that feeding CD leads to both obesity and behavioural changes in rats. Overall, these effects were more pronounced in older rats, with the behavioural effects being particularly gender dependent.

The impact of maternal cafeteria diet on anxiety-related behaviour and exploration in the offspring.

Contemporary trends in obesity mean that research into whether unbalanced diets could impact on behavioural traits became increasingly important. The timing of exposure to obesity is particularly important, as sensitive periods during development have been identified where dietary extremes play a critical role in determining adult risk of physiological dysfunction. To this end, female Wistar rats were fed on chow or cafeteria diet (CD) for 8 weeks from weaning until mating. Half of the mated animals within each group were crossed-over to the alternative diet. This generated four treatment groups, differing in their pre-gestational and gestational diets. After birth, offspring of dams from each of the 4 pregnancy groups were further divided into groups, either being fed chow or CD throughout lactation. Anxiety-related behaviour and exploration in the offspring were tested in the Elevated Plus Maze (EMP) and the Open Field (OF) at 10 weeks of age. Maternal obesity significantly reduced the EPM locomotor activity in male and female offspring and grooming in males. Lactational CD had an anxiolytic effect in male offspring as shown in the EPM (increased entries into and more time on open arms) and the OF (shorter latency to enter the centre). In both sexes, lactational CD reduced grooming upon exposure to the EPM and the OF. Post mortem analysis revealed a stimulant effect of lactational CD on adipose tissue growth. The present study demonstrates that pre-gestational, gestational and lactational maternal CD programme behaviour in the offspring with lactational CD reducing anxiety in the male offspring.

Exposure to maternal consumption of cafeteria diet during the lactation period programmes feeding behaviour in the rat.

Lactational overfeeding programmes obesity in the adult rat, and also impacts on adult emotional behaviour. The present study investigated the impact of exposing the lactating female to a hypercaloric diet on structural aspects of feeding behaviour in the adult offspring as measured by the behavioural satiety sequence (BSS). Lactating Wistar rats were fed a hypercaloric cafeteria diet (CD) in addition to chow. Controls were fed on chow only. All offspring were chow fed after weaning. BSS was tested in 12-15 week old offspring. At 20 weeks of age, monoamine neurotransmitter levels were measured in selected brain regions. When exposed to a palatable 1-h test meal, offspring responded with the same latency to feed, regardless of lactational diet. Total food intake during the test was unaffected by lactational diet. Control offspring showed a normal BSS pattern. Male CD offspring displayed shorter feeding bouts (P<0.05) with an overall higher bout frequency (P<0.001) and their latency to rest was delayed (P<0.001). Overall eating frequency (P<0.05), but not duration was increased in male CD offspring. Although the transition from feeding to resting was not affected by lactational CD, CD males fed for longer at the beginning of the test meal and were more active towards the end. CD females displayed an increased number of feeding bouts (P<0.05) and they spent more time eating (P<0.05). Resting latency was delayed (P<0.05) and overall time spent resting was shortened (P<0.01). Frequency of eating was increased in the middle of the test meal. The onset of satiety as indicated by the transition point between eating and resting was delayed in CD females (P<0.001). In both sexes, hypothalamic 5-hydroxytryptamine (5-HT) was increased (P<0.05 in females, P<0.01 in males) and 5-HT turnover was reduced by lactational CD (P<0.001 in females, P<0.01 in males). Lactational CD led also to an increase in dopamine (DA) (P<0.01). Hypothalamic DA metabolism (DOPAC+HVA/DA ratio) was overall lower in females than in males (P<0.01). This study indicates a programming effect of lactational CD on feeding behaviour and brain monoaminergic neurons.

5-HT(1A)-receptor over-expressing mice: genotype and sex dependent responses to antidepressants in the forced swim-test.

Deficiencies in serotonergic neurotransmission are involved in the pathophysiology of depression. Due to its modulatory effect on serotonin (5-HT) release, the 5-HT(1A)-receptor is thought to play a decisive role in the therapy of this mood disorder. However, it is not fully understood how antidepressant effects are mediated by pre- and postsynaptic receptor sites. In this study we examined the impact of postsynaptic 5-HT(1A)-receptor over-expression in corticolimbic areas of male and female mice on the performance in the forced swim-test (FST). Furthermore, we investigated their response to the serotonin selective reuptake inhibitor (SSRI) citalopram in comparison to the selective noradrenaline reuptake inhibitor reboxetine, as well as the partial 5-HT(1A)-receptor agonists, buspirone and S 15535. Additionally, these drugs were evaluated in the open field-test in order to observe effects on motor activity. The density of 5-HT(1A)-receptors in discrete corticolimbic regions was determined in detail by quantitative autoradiography with [(3)H]8-OH-DPAT to investigate genotype as well as sex dependent differences in the expression pattern. [(3)H]8-OH-DPAT binding differed depending on sex with female mice of both genotypes displaying higher receptor binding in distinct brain areas. In the FST untreated male but not female over-expressing (OE) mice showed an antidepressant-like behaviour compared to wild-type (WT) mice. Citalopram yielded an antidepressant effect without influencing locomotor activity in OE mice but not in WT mice. Reboxetine had no antidepressant-like effect in OE mice, but sex-dependently in WT mice. The two partial agonists, buspirone and S 15535 produced no antidepressant-like activity in both genotypes and sexes, but aberrant motor effects. The antidepressant-like phenotype of male transgenic mice accounts for an involvement of postsynaptic 5-HT(1A)-receptors in the FST behaviour. In addition, the selective over-expression of postsynaptic 5-HT(1A)-receptors in mice contributes to the antidepressant response to citalopram in the FST. Although further pharmacological analysis is required, the data provide novel support for a role of postsynaptic 5-HT(1A)-receptors in the effects of SSRIs.