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Wolfram syndrome is a rare autosomal recessive disorder caused by mutations in the wolframin ER transmembrane glycoprotein (WFS1) gene and characterized by diabetes mellitus, diabetes insipidus, optic atrophy and deafness. In experimental models the homozygous Wfs1 mutant mice have a full penetrance and clearly expressed phenotype, whereas heterozygous mutants have a less-pronounced phenotype between the wild-type and homozygous mutant mice. Heterozygous WFS1 mutations have been shown to be significant risk factors for diabetes and metabolic disorders in humans. In the present study we analyzed the response of heterozygous Wfs1 mice to high fat diet (HFD) by exploring potential outcomes and molecular changes induced by this challenge. The HFD treatment increased the body weight (BW) similarly both in Wfs1 wild-type (WT) and heterozygous (HZ) mice, and therefore HFD also prevented the impaired BW gain found in Wfs1 mutant mice. In Wfs1HZ mutant mice, HFD impaired the normalized insulin secretion and the expression of ER stress genes in isolated pancreatic islets. These results suggest that Wfs1HZ mice have a decreased insulin response and pronounced cellular stress response due to a higher sensitivity to HFD as hypothesized. In Wfs1HZ mice, HFD increased the expression of Ire1α and Chop in pancreas and decreased that of Ire1α and Atf4 in liver. The present study shows that HFD can disturb insulin function with an increased ER stress in Wfs1HZ mice and only one functional Wfs1 gene copy is not sufficient to compensate this challenge. In conclusion, our study indicates that quantitative Wfs1 gene deficiency is sufficient to predispose the carriers of single functional Wfs1 copy to diabetes and metabolic syndrome and makes them susceptible to the environmental challenges such as HFD.

There are relatively few studies surveying how meteorological and qualitative characteristics of tree sites in urban areas influence Hymenoscyphus fraxineus (T. Kowalski) Baral et al. ( Chalara fraxinea T. Kowalski, = Hymenoscyphus pseudoalbidus Queloz et al.) or infection by that. Based on the significance of ash trees in urban areas and the frequent use of ash species and their varieties, it becomes increasingly important to identify factors that drive the pathogen. The aim of this study was to identify the effects of both meteorological parameters and tree characteristics on the severity of the disease in the city of Leipzig, Germany. We assessed 401 ash trees along streets and in green spaces distributed across Leipzig. The assessment included scoring the level of damage by H. fraxineus, visually estimating the degree of soil sealing under a tree's crown, measuring tree height, the height of crown base and the diameter at breast height, determining sex, year of planting and species variety, as well as recording qualitative characteristics (e.g. secondary pathogens, feeding marks). Mean relative air humidity, mean annual temperature and a combined index of meteorological conditions were used as meteorological parameters. In addition, we sampled tissue of 45 ash trees for the purpose of genetically verifying the occurrence of H. fraxineus. We analysed the effects of all variables on ash dieback by using linear mixed effect models. Results show an extensive damage by H. fraxineus in the whole city area with 70% of trees being infected. Mean relative air humidity and the index of meteorological conditions show a significant positive influence on the severity of ash dieback, whereas year of planting has a negative effect. Trees with feeding marks at the trunk tend to be more damaged by the fungus. Moreover, we found a significant difference in susceptibility between F. excelsior and its variety 'Atlas'. Both meteorological conditions and the susceptibility of species

Im Jahr 2016 wurde im Stadtgebiet Leipzig das Vorkommen des Eschentriebsterbens an Stadtbäumenerfasst, um zu untersuchen, welche Ursachen dessen Auftreten erklären könnten.

It has been shown that mutations in the WFS1 gene make humans more susceptible to mood disorders. Besides that, mood disorders are associated with alterations in the activity of serotonergic and noradrenergic systems. Therefore, in this study, the effects of imipramine, an inhibitor of serotonin (5-HT) and noradrenaline (NA) reuptake, and paroxetine, a selective inhibitor of 5-HT reuptake, were studied in tests of behavioral despair. The tail suspension test (TST) and forced swimming test (FST) were performed in Wfs1-deficient mice. Simultaneously, gene expression and monoamine metabolism studies were conducted to evaluate changes in 5-HT- and NA-ergic systems of Wfs1-deficient mice. The basal immobility time of Wfs1-deficient mice in TST and FST did not differ from that of their wild-type littermates. However, a significant reduction of immobility time in response to lower doses of imipramine and paroxetine was observed in homozygous Wfs1-deficient mice, but not in their wild-type littermates. In gene expression studies, the levels of 5-HT transporter (SERT) were significantly reduced in the pons of homozygous animals. Monoamine metabolism was assayed separately in the dorsal and ventral striatum of naive mice and mice exposed for 30 min to brightly lit motility boxes. We found that this aversive challenge caused a significant increase in the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, in the ventral and dorsal striatum of wild-type mice, but not in their homozygous littermates. Taken together, the blunted 5-HT metabolism and reduced levels of SERT are a likely reason for the elevated sensitivity of these mice to the action of imipramine and paroxetine. These changes in the pharmacological and neurochemical phenotype of Wfs1-deficient mice may help to explain the increased susceptibility of Wolfram syndrome patients to depressive states.

In this behavioural and pharmacological study in male CCK2 receptor-deficient mice (CCK2−/−), we evaluated the role of the interaction of endocannabinoids (eCBs) and cholecystokinin (CCK) on the regulation of anxiety-related and motor behaviours. Repeated treatment with amphetamine (2 mg/kg daily for four days) induced slightly weaker motor sensitisation in CCK2−/− mice compared to their wild-type (CCK2+/+) littermates. Co-administration of rimonabant (1 mg/kg) with amphetamine antagonised the development of motor sensitisation in CCK2+/+ mice. However, we did not find a similar effect of rimonabant in CCK2−/− mice. We did not find any differences between the behaviour of CCK2+/+ and CCK2−/− mice in models designed to assess emotional behaviours (dark/light exploration, marble burying and conditioned place aversion). This study supports the hypothesis that eCBs play a role in the development of amphetamine-induced sensitisation. Moreover, we have demonstrated that intact CCK2 receptors are necessary for the development of eCB-mediated sensitisation to amphetamine.

Aim Late Pleistocene glacial changes had a major impact on many boreal and temperate taxa, and this impact can still be detected in the present-day phylogeographic structure of these taxa. However, only minor effects are expected in species with generalist habitat requirements and high dispersal capability. One such species is the white-tailed eagle, Haliaeetus albicilla, and we therefore tested for the expected weak population structure at a continental level in this species. This also allowed us to describe phylogeographic patterns, and to deduce Ice Age refugia and patterns of postglacial recolonization of Eurasia. Location Breeding populations from the easternmost Nearctic (Greenland) and across the Palaearctic (Iceland, continental Europe, central and eastern Asia, and Japan). Methods Sequencing of a 500 base-pair fragment of the mitochondrial DNA control region in 237 samples from throughout the distribution range. Results Our analysis revealed pronounced phylogeographic structure. Overall, low genetic variability was observed across the entire range. Haplotypes clustered in two distinct haplogroups with a predominantly eastern or western distribution, and extensive overlap in Europe. These two major lineages diverged during the late Pleistocene. The eastern haplogroup showed a pattern of rapid population expansion and colonization of Eurasia around the end of the Pleistocene. The western haplogroup had lower diversity and was absent from the populations in eastern Asia. These results suggest survival during the last glaciation in two refugia, probably located in central and western Eurasia, followed by postglacial population expansion and admixture. Relatively high genetic diversity was observed in northern regions that were ice-covered during the last glacial maximum. This, and phylogenetic relationships between haplotypes encountered in the north, indicates substantial population expansion at high latitudes. Areas of glacial meltwater runoff and proglacial

Most of the white-tailed eagle (Haliaeetus albicilla) populations in Europe experienced dramatic declines during the twentieth century. However, owing to intense conservation actions and the ban of DDT and other persistent pollutants, populations are currently recovering. We show that despite passing through demographic bottlenecks, white-tailed eagle populations have retained significant levels of genetic diversity. Both genetic and ringing data indicate that migration between populations has not been a major factor for the maintenance of genetic variability. We argue that the long generation time of eagles has acted as an intrinsic buffer against loss of genetic diversity, leading to a shorter effective time of the experienced bottleneck. Notably, conservation actions taken in several small sub-populations have ensured the preservation of a larger proportion of the total genetic diversity than if conservation had focused on the population stronghold in Norway. For conservation programmes targeting other endangered, long-lived species, our results highlight the possibility for local retention of high genetic diversity in isolated remnant populations.

15th International Symposium on Regulatory Peptides

Rationale Evidence suggests that GABA and CCK have opposite roles in the regulation of anxiety. Objective The aim of the present work was to study diazepam-induced anxiolytic-like action and impairment of motor co-ordination, and the parameters of benzodiazepine receptors in mice lacking CCK2 receptors. Methods The action of diazepam (0.5–3 mg/kg IP) was studied in the elevated plus-maze model of anxiety and rotarod test using mice lacking CCK2 receptors. The parameters of benzodiazepine receptors were analysed using [3H]-flunitrazepam binding. Results In the plus-maze test, the exploratory activity of the homozygous (−/−) mice was significantly higher compared to their wild-type (+/+) littermates. However, the wild-type (+/+) mice displayed higher sensitivity to the anxiolytic-like action of diazepam. Even the lowest dose of diazepam (0.5 mg/kg) induced a significant increase of open arm entries in the wild-type (+/+) mice. A similar effect in the homozygous (−/−) mice was established after the administration of diazepam 1 mg/kg. The highest dose of diazepam (3 mg/kg) caused a prominent anxiolytic-like effect in the wild-type (+/+) mice, whereas in the homozygous (−/−) animals suppression of locomotor activity was evident. The performance of the homozygous (−/−) mice in the rotarod test did not differ from that of the wild-type (+/+) littermates. However, a difference between the wild-type (+/+) and homozygous (−/−) animals became evident after treatment with diazepam. Diazepam (0.5 and 3 mg/kg) induced significantly stronger impairment of motor co-ordination in the homozygous (−/−) mice compared to their wild-type (+/+) littermates. The density of benzodiazepine binding sites was increased in the cerebellum, but not in the cerebral cortex and hippocampus, of the homozygous (−/−) mice. Conclusions Female mice lacking CCK2 receptors are less anxious than their wild-type (+/+) littermates. The reduced anxiety in homozygous (−/−) mice probably explains why the adminis

Poster session P.3. Anxiety disorder and anxiolytic

No abstract available

Rationale: Cholecystokinin (CCK) has been shown to coexist and interact with dopamine in the regulation of behaviour. Two different CCK receptors (CCK1 and CCK2) have an opposite influence on the activity of dopamine neurons. Stimulation of CCK2 receptors decreases the release of dopamine and that receptor could mediate the neuroleptic-like effect of CCK. Objective: To investigate the activity of the dopaminergic system in pharmacological experiments on CCK2 receptor (CCK2R)-deficient mice. Methods: We used age- and sex-matched littermates in all our experiments. To evaluate the behavioural differences, we performed the rotarod test and measured the locomotor activity of animals using computer-connected photoelectric motility boxes. Amphetamine and apomorphine, two dopaminergic drugs with different pharmacodynamic properties, were used to influence the activity of the dopaminergic system in the brain. Neurochemical differences related to the different genotype were analysed by means of high-performance liquid chromatography and radioligand binding studies. Results: Motor co-ordination was significantly impaired in the rotarod test of CCK2R receptor-deficient mice. Moreover, the locomotor activity of heterozygous (+/–) and homozygous (–/–) CCK2R receptor-deficient mice was somewhat reduced. A low dose of apomorphine (0.1 mg/kg), an unselective agonist of dopamine receptors, suppressed locomotor activity significantly more in homozygous (–/–) and heterozygous (+/–) mutant mice than in their wild-type (+/+) littermates. Amphetamine (3–6 mg/kg), increasing release of dopamine from the presynaptic terminals, caused a dose-dependent motor stimulation in wild-type (+/+) mice. In heterozygous (+/–) and homozygous (–/–) mice, a lower dose of amphetamine (3 mg/kg) did not alter the locomotor activity, whereas the higher dose of (6 mg/kg) induced a significantly stronger increase in locomotor activity in homozygous (–/–) mice than in their heterozygous (+/–) and wild-type (+/+

Apomorphine-induced behavioural sensitization was studied in male Wistar rats. The acute administration of apomorphine (0.5 mg/kg s.c.), a dopamine agonist, did not affect the locomotor activity of rats, but it caused stereotyped behaviour characterized by repeated gnawing, licking and sniffing. A significant increase in the locomotor activity became evident after repeated treatments with apomorphine (0.5 mg/kg twice daily for 14 days). However, there were marked individual differences in the sensitization of rats to apomorphine. One third of animals did not react with increased locomotor activity even after the 2-week administration of apomorphine, whereas the other one third needed only a few injections to display increased behavioural response to apomorphine. The behavioural response of the remaining one third of rats was between weak and strong responders. Simultaneously, the stereotyped behaviour occurred earlier and its intensity tended to be lower after repeated treatment with apomorphine. Nevertheless, the established changes of stereotyped behaviour did not correlate with the increase of locomotor activity. The administration of amphetamine (2.5 mg/kg, s.c.), an indirect dopamine agonist, but not a non-competitive NMDA antagonist dizocilpine (0.25 mg/kg i.p.), tended to cause a similar response profile with apomorphine in sensitized rats. The ED50 values of the dopamine antagonists blocking apomorphine-induced increase in the locomotor activity were the following: 0.09 mg/kg for raclopride (dopamine D2 antagonist), 0.023 mg/kg for SCH 23390 (dopamine D1 antagonist), 6.42 mg/kg for clozapine (dopamine D4 antagonist). This supports the involvement of D1 and D2 receptors in the expression of apomorphine-induced behavioural sensitization. The concomitant administration of dizocilpine (0.5 mg/kg), SCH 23390 (0.05 mg/kg), raclopride (0.1 mg/kg) and clozapine (20 mg/kg) with apomorphine (0.5 mg/kg twice daily for 2 weeks) antagonized the development of behavioural

This study investigated the interplay of cholecystokinin (CCK) and endogenous opioid peptides in the regulation of anxiety. The acute administration of non-selective CCK agonist caerulein (1 and 5 μg/kg) and a selective CCKB receptor agonist BOC-CCK-4 (1, 10 and 50 μg/kg) induced a dose-dependent anxiogenic-like action in the plus-maze model of anxiety. BOC-CCK-4 displayed a similar efficacy with caerulein, indicating that the described effect was mediated via CCKB receptor subtype. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity of rats in the plus-maze. However, the combination of naloxone with the sub-effective doses of caerulein (1 μg/kg) and BOC-CCK-4 (1 μg/kg) induced a significant inhibition of exploratory behaviour in rats. Accordingly, CCK and endogenous opioid peptides have an antagonistic role in the exploratory model of anxiety in rats.

The involvement of nitrergic mechanisms in the behavioural effects of diazepam in rats was studied in the elevated plus-maze, open-field and rotarod tests. Administration of the nitric oxide (NO) precursor l-arginine (100 mg/kg, i.p.), assumed to increase the synthesis of NO, abolished the anxiolytic-like effect of diazepam (2 mg/kg, i.p.) in the elevated plus-maze, whereas the inactive enantiomer d-arginine (100 mg/kg) did not. Neither diazepam alone nor in combination with l- or d-arginine affected the exploratory activity of animals in the open field. Pretreatment with l-arginine (100 and 200 mg/kg) did not modify the motor impairment of rats after diazepam (3 mg/kg) in the rotarod test. Diazepam (2 mg/kg i.p.) did not inhibit the cortical or hippocampal cytosolic NO synthase activity measured ex vivo by [3H]l-arginine assay. Diazepam was similarly ineffective in in vitro studies at concentrations up to 10 μM. We conclude that a suppression of NO synthase activity may be important in the anxiolytic-like effect of benzodiazepines. However, diazepam does not inhibit NO synthase directly, but may affect NO synthase activity indirectly via some unknown mechanism.

Forty-five male Wistar rats were selected according to their behavior in the elevated plus-maze. They were separated as follows: animals with low exploratory activity (`anxious'), an `intermediate' group and animals having high exploratory activity (`non-anxious'). Various receptor binding studies and hormonal assays were also performed in these selected rats. The affinity of 5-hydroxytryptamine 5-HT2A receptors in the frontal cortex was lower in the `anxious' rats compared to home-cage controls and `non-anxious' animals. Moreover, the number of cholecystokinin (CCK) receptors in the hippocampus was significantly elevated in the `anxious' group compared to home-cage control animals. The blood levels of growth hormone (GH) were significantly lower in the `non-anxious' rats compared to `anxious' counterparts. In conclusion, it seems likely that the decreased exploratory activity of rats is related to the increased 5-hydroxytryptamine (5-HT) and CCK mediated neurotransmission in the brain. The different serum levels of GH in the selected rats probably reflect alterations in the activity of 5-HT and CCK.

No abstract available

Caerulein, a non-selective agonist of cholecystokinin (CCK) receptors, is shown to suppress locomotor activity in rodents via stimulation of CCKA receptors. In the present study we examined the possible involvement of nitric oxide (NO) in caerulein-induced hypolocomotion in rats. Caerulein (10 μg/kg) markedly decreased the horizontal and vertical components of locomotor activity in rats measured in dark motility boxes. Pretreatment with a nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME), at 5 mg/kg i.p., abolished the inhibiting action of caerulein on the horizontal activity, but did not affect the reduced frequency of rearing. The other doses of l-NAME (1, 10 and 20 mg/kg) were ineffective against caerulein. As L-NAME at this dose range does not stimulate locomotor activity, it is likely that NO is involved in the motor suppressant effect of systemically administered caerulein.

No abstract available

The action of inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME) (1-20 mg kg-1) on the exploratory behaviour of rats in the elevated plus-maze was studied. L-NAME induced an anxiolytic-like effect in the plus-maze test, showing a reverse U-shape action behaviour, with a maximal effect at 10 mg kg-1. This effect was not related to a non-specific increase in motor activity, since in the open field test L-NAME did not affect locomotor activity of rats. Pretreatment of rats with L-NAME (1-10 mg kg-1) also tended to attenuate the anti-exploratory action of CCK agonist caerulein (5 micrograms kg-1), but this action was not significant. In conclusion, it appears that NO may be involved in the process that can lead to anxiety in the rat.

We studied the aggressive behaviour induced by repeated treatment with apomorphine, a dopamine agonist (0.5 mg/kg s.c. twice daily, 10 days), in rats. The first signs of defensive aggressiveness appeared on the third day of apomorphine treatment and were generally seen on the 7th day. Aggressiveness induced by a challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day was antagonized by haloperidol (0.05 and 0.1 mg/kg i.p.) and clozapine (10 mg/kg i.p.). An antagonist of N-methyl-D-aspartate (NMDA)-gated channels, dizocilpine (MK-801), also blocked the aggressive behaviour at 0.25 and 0.5 mg/kg i.p. but caused ataxia. When dizocilpine (0.25 mg/kg i.p.) and apomorphine were coadministered for 10 days, aggressive behaviour did not develop. At 0.025 mg/kg i.p., dizocilpine even accelerated the appearance of apomorphine-induced aggressive behaviour, which manifested on the 3rd day in all rats. In a separate study, a 7-day treatment with dizocilpine (0.25–1 mg/kg i.p.) of rats, sensitized by a prior 10-day apomorphine treatment, did not reverse the established aggressive behaviour. The coadministration of apomorphine and cholecystokinin (CCK)-A or -B antagonists, devazepide or L-365,260 (0.01–2.5 mg/kg i.p.) respectively, neither affected development of apomorphine-induced aggressive behaviour nor intensity of aggressiveness in the sensitized rats. In binding studies neither density nor affinity of striatal dopamine D2 receptors was changed by acute or chronic apomorphine treatment. The number of [3H]pCCK-8 binding sites in the frontal cortex increased already after a single injection of apomorphine. After 10-day administration of apomorphine, a significant upregulation of [3H]pCCK-8 binding sites occurred in the frontal cortex and striatum, but a downregulation was observed in the hippocampus. A challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day of experiment, normalized the upregulated CCK receptors in the frontal cortex and striatum. Acute apomorphin

We compared the effects of unselective cholecystokinin (CCK) agonists (caerulein and CCK-8s) and a CCKB agonist CCK-4 on the secretion of thyrotropin (TSH), growth hormone (GH) and prolactin (PRL) in male rats. The subcutaneous (s.c.) administration of caerulein and CCK-8s suppressed dose-dependently TSH and GH levels. In contrast, when given into the 3rd brain ventricle (i.c.v.) caerulein dose-dependently elevated the GH levels. Next the importance of the afferent vagal nerves was studied in the action of caerulein and CCK-4. Subdiaphragmatic vagotomy itself decreased cold-stimulated TSH levels but abolished the suppressing effect of intraperitoneal (i.p.), and apparently also that of the i.c.v. caerulein. GH and PRL levels were altered neither by vagotomy nor caerulein. CCK-4 did not affect hormone levels. Atropine and butylscopolamine (i.p.) themselves did not alter TSH, PRL or GH secretion in intact rats. Neither did they reverse the effect of caerulein on TSH. In conclusion, CCKA receptors dominate in TSH and CCKB receptors in GH regulation. CCKA receptors in the gastrointestinal tract, related to the nervus vagus are mediating the inhibitory effect of caerulein upon TSH secretion but inhibition of GH secretion does not depend on the nervus vagus. CCKB receptors in the brain stem or near the 3rd brain ventricle are responsible for stimulation of GH secretion.

No abstract available

We compared the action of subdiaphragmatic vagotomy upon the anti-exploratory and motor depressant effects of caerulein, an agonist of cholecystokinin (CCK) receptors, in male rats. Vagotomized rats entered more frequently into the open arms of elevated plus-maze compared to intact control rats. Caerulein (1 μg/kg subcutaneously (s.c.)) significantly suppressed the exploratory behaviour in vagotomized rats but not in intact and sham-operated rats. In contrast, subdiaphragmatic vagotomy did not change the locomotor activity of rats in open field compared to intact and sham-operated animals. At a higher dose (10 μg/kg s.c.), the caerulein pretreatment markedly decreased the number of line crossings, rearings and head-dippings of intact animals in open field. In sham-operated rats caerulein also suppressed the locomotor activity, whereas in vagotomized rats it only tended to reduce the frequency of rearings. Consequently, the present study revealed the different action of vagotomy upon the motor depressant and anti-exploratory effects of caerulein. These results support the view that CCKA receptors in the gastrointestinal tract are mediating the motor depressant, whereas CCKB receptors in the brainstem are involved into the mediation of anti-exploratory effect of caerulein.

(NMDA, 20 mg/kg) produced a clear decrease in mouse exploration in open parts of an elevated plus-maze. Paradoxically, 40 mg/kg NMDA did not modify the behavior of the mice in the plus-maze. NMDA at a dose of 80 mg/kg again depressed the exploratory activity of mice, but this effect was accompanied with tremor and compulsive tail biting. The ‘anti-exploratory’ dose of NMDA (20 mg/kg) increased, whereas the ‘tremorigenic’ dose (80 mg/kg) significantly decreased the number of cholecystokinin (CCK) binding sites in the mouse cerebral cortex. The competitive NMDA antagonist (±)-CPP (2.5–5 mg/kg) and the non-competitive antagonist MK-801 (0.25 mg/kg) antagonized the anti-exploratory effect of NMDA (20 mg/kg). The tricyclic antidepressant imipramine (5 mg/kg, but not 1 or 10 mg/kg) also attenuated the inhibition of exploratory activity induced by NMDA. Of three CCK receptor antagonists tested, the unselective CCK antagonist proglumide (1 mg/kg, but not 0.1 and 10 mg/kg) significantly opposed the anti-exploratory action of NMDA. The selective CCK antagonists L-365,260 (1 μg/kg) and devazepide (1 μg/kg) were evidently weaker antagonists of NMDA. Furthermore, 10 μg/kg of L-365,260, a CCK-B receptor antagonist, and 1 mg/kg of devazepide, a CCK-A receptor antagonist, even tended to augment the effect of NMDA in the plus-maze. The results of the present study seem to give some support to the notion that not only NMDA receptors, but also CCK-ergic mechanisms are involved in the modulation of anti-exploratory action of NMDA in the elevated plus-maze.