Joining forces: the need to combine science and ethics to address problems of validity and translation in neuropsychiatry research using animal models.

BACKGROUND: Current policies regulating the use of animals for scientific purposes are based on balancing between potential gain of knowledge and suffering of animals used in experimentation. The balancing process is complicated, on the one hand by plurality of views on our duties towards animals, and on the other hand by more recent discussions on uncertainty in the probability of reaching the final aim of the research and problems of translational failure. METHODS: The study combines ethical analysis based on a literature review with neuropsychiatry-related preclinical research as a case study. RESULTS: Based on the analysis and the case study we show that neuropsychiatry-related preclinical research is an especially interesting case from an ethical perspective. The 3R principles (Replacement, Reduction and Refinement) are used to minimize the negative consequences for the animals used in research. However, neuropsychiatric research is characterized by specific challenges in assessing the probability of success of reaching the final aim, due to our limited mechanistic knowledge of human neuropsychiatric illness. Consequently, the translational value of the currently used animal models may be difficult to prove, which undermines the validity of these models and complicated the ethical assessment. CONCLUSIONS: We conclude that a combined approach that deals with both science and the ethical dimensions is necessary to address the problems of validity and translation in neuropsychiatry-related preclinical research. We suggest this approach to comprise first, improved experimental methods, e.g. by using systematic reviews, second, a more patients-based approach that leads to models that reflect interindividual variation better, and third, more interdisciplinary cooperation.

Brief Report: Association Between Autism Spectrum Disorder, Gastrointestinal Problems and Perinatal Risk Factors Within Sibling Pairs.

Autism spectrum disorder (ASD) has been associated with gastrointestinal (GI) problems, but the nature of this association is unclear. Parents to siblings, concordant or discordant for ASD (N = 217), participated in a web survey covering mother's weight gain during pregnancy, maternal viral/bacterial infection and use of antibiotics, duration of breastfeeding, mode of delivery, birth weight and child GI problems. ASD was associated with GI problems and perinatal environmental risk, based on a summation of maternal infection and antibiotic use during pregnancy and/or the breastfeeding period. The association between GI problems and ASD remained within the sibling pairs (ß = 1.23; p < .001) in the adjusted model. Our results indicate non-shared environmental effects on the ASD/GI association, but none of the factors examined explained the link.

Multilevel control of glucose homeostasis by adenylyl cyclase 8.

AIMS/HYPOTHESIS: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an important role in glucose-induced signalling and glucose homeostasis. METHODS: We used pharmacological and genetic approaches in beta cells to determine secretion and calcium metabolism. Furthermore, Adcy8 knockout mice were characterised. RESULTS: In clonal beta cells, inhibitors of adenylyl cyclases or their downstream targets reduced the glucose-induced increase in cytosolic calcium and insulin secretion. This was reproduced by knock-down of ADCY8, but not of ADCY1. These agents also inhibited glucose-induced increase in cytosolic calcium and electrical activity in primary beta cells and similar effects were observed after ADCY8 knock-down. Moreover, insulin secretion was diminished in islets from Adcy8 knockout mice. These mice were glucose intolerant after oral or intraperitoneal administration of glucose whereas their levels of glucagon-like peptide-1 remained unaltered. Finally, we knocked down ADCY8 in the ventromedial hypothalamus to evaluate the need for ADCY8 in the central regulation of glucose homeostasis. Whereas mice fed a standard diet had normal glucose levels, high-fat diet exacerbated glucose intolerance and knock-down mice were incapable of raising their plasma insulin levels. Finally we confirmed that ADCY8 is expressed in human islets. CONCLUSIONS/INTERPRETATIONS: Collectively, our findings demonstrate that ADCY8 is required for the physiological activation of glucose-induced signalling pathways in beta cells, for glucose tolerance and for hypothalamic adaptation to a high-fat diet via regulation of islet insulin secretion.

Longitudinal changes in the physical activity of adolescents with anorexia nervosa and their influence on body composition and leptin serum levels after recovery.

OBJECTIVE: Patients with anorexia nervosa (AN) are often observed to have high levels of physical activity, which do not necessarily diminish after a successful therapy. Previous studies have shown that body fat tissue recovery in these patients is associated with a disproportional restoration of the adipocyte hormone, leptin. Therefore, we wondered whether the individual variation in physical activity in AN patients prior to treatment may be related to body fat percentage and plasma leptin level outcome. METHOD: Body fat percentage, leptin serum, and physical activity levels (accelerometer) were measured in adolescents with an (n=37, age 13 to 17.5 years) at initial assessment, at the end of study participation (median 12 months), and at one-year follow-up. RESULTS: Accelerometer data were used to split the patients in two groups: those with low (n=26) and those with high levels of physical activity (HLPA, n=11). These groups did not differ in terms of age, IQ, presence of menses, BMI and season of admission. The HLPA group was characterized by a longer total duration of illness. Physical activity levels during therapy decreased for the group with initially HLPA and increased for the group with low levels of physical activity (to comparable levels). Physical activity remained stable after one year. The increase in body fat percentage and leptin levels were dependent on the recovery status; however, recovered patients with initially HLPA had significantly higher fat mass during the follow-up. DISCUSSION: HLPA, an important modulator of AN progression in adolescents, can be successfully diminished by therapeutic intervention. Among recovered patients, those with initially HLPA had higher fat mass levels than those with low levels of physical activity. This finding suggests that HLPA are an important modulator of the body composition recovery mechanism.

The expression of excessive exercise co-segregates with the risk of developing an eating disorder in women.

Excessive exercise (EE) is an important symptom of eating disorders (ED) and is a likely risk factor for developing ED, however, no population-based studies have been performed on the relationship between EE and obtaining ED diagnosis. The aim of this study was to examine the co-occurrence of EE and ED diagnosis in a general population of women. Data for 778 females (age min=30, max=55) from the Saint Thomas Twin Registry, London were used. Phenotypes analyzed included self-reported time spent on physical activity per week, ED diagnosis, Eating Disorder Inventory results (EDI-III), age, BMI and kinship (twin pair). Generalized Estimating Equation analysis showed that only EE (>5 h of exercise per week) and Bulimia Subscale of EDI-III were significantly associated with obtaining ED diagnosis throughout the life. These data revealed that the odds of ever being diagnosed with an ED are more than 2.5 times higher for excessive exercisers compared to individuals with lower activity levels. These data support the notion that EE may be an important risk factor for developing an ED in women.

New operant model of reinstatement of food-seeking behavior in mice.

RATIONALE: A major problem in treating obesity is the high rate of relapse to abnormal food-taking behavior when maintaining diet. OBJECTIVES: The present study evaluates the reinstatement of extinguished palatable food-seeking behavior induced by cues previously associated with the palatable food, re-exposure to this food, or stress. The participation of the opioid and dopamine mechanisms in the acquisition, extinction, and cue-induced reinstatement was also investigated. MATERIALS AND METHODS: C57BL/6 mice were first trained on a fixed-ratio-1 schedule of reinforcement to obtain chocolate-flavored pellets during 20 days, which was associated to a stimulus light. Operant behavior was then extinguished during 20 daily sessions. mRNA levels of opioid peptide precursors and dopamine receptors were evaluated in the brain by in situ hybridization and RT-PCR techniques. RESULTS: A reinstatement of food-seeking behavior was only obtained after exposure to the food-associated cue. A down-regulation of prodynorphin mRNA was found in the dorsal striatum and nucleus accumbens after the acquisition, extinction, and reinstatement of the operant behavior. Extinction and reinstatement of this operant response enhanced proenkephalin mRNA in the dorsal striatum and/or the nucleus accumbens core. Down-regulation of D2 receptor expression was observed in the dorsal striatum and nucleus accumbens after reinstatement. An up-regulation of PDYN mRNA expression was found in the hypothalamus after extinction and reinstatement. CONCLUSIONS: This study provides a new operant model in mice for the evaluation of food-taking behavior and reveals specific changes in the dopamine and opioid system associated to the behavioral responses directed to obtain a natural reward.

The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum.

BACKGROUND: Various drugs of abuse activate intracellular pathways in the brain reward system. These pathways regulate the expression of genes that are essential to the development of addiction. To reveal genes common and distinct for different classes of drugs of abuse, we compared the effects of nicotine, ethanol, cocaine, morphine, heroin and methamphetamine on gene expression profiles in the mouse striatum. RESULTS: We applied whole-genome microarray profiling to evaluate detailed time-courses (1, 2, 4 and 8 hours) of transcriptome alterations following acute drug administration in mice. We identified 42 drug-responsive genes that were segregated into two main transcriptional modules. The first module consisted of activity-dependent transcripts (including Fos and Npas4), which are induced by psychostimulants and opioids. The second group of genes (including Fkbp5 and S3-12), which are controlled, in part, by the release of steroid hormones, was strongly activated by ethanol and opioids. Using pharmacological tools, we were able to inhibit the induction of particular modules of drug-related genomic profiles. We selected a subset of genes for validation by in situ hybridization and quantitative PCR. We also showed that knockdown of the drug-responsive genes Sgk1 and Tsc22d3 resulted in alterations to dendritic spines in mice, possibly reflecting an altered potential for plastic changes. CONCLUSIONS: Our study identified modules of drug-induced genes that share functional relationships. These genes may play a critical role in the early stages of addiction.

Interspecies trait genetics reveals association of Adcy8 with mouse avoidance behavior and a human mood disorder.

BACKGROUND: Identifying susceptibility genes for endophenotypes by studying analogous behaviors across species is an important strategy for understanding the pathophysiology underlying psychiatric disorders. This approach provides novel biological pathways plus validated animal models critical for selective drug development. One such endophenotype is avoidance behavior. METHODS: In the present study, novel automated registration methods for longitudinal behavioral assessment in home cages are used to screen a panel of recently generated mouse chromosome substitution strains that are very powerful in quantitative trait loci (QTL) detection of complex traits. In this way, we identified chromosomes regulating avoidance behavior (increased sheltering preference) independent of motor activity levels (horizontal distance moved). Genetic information from the mouse QTL-interval was integrated with that from the homologous human linkage region for a mood disorder. RESULTS: We genetically mapped a QTL for avoidance behavior on mouse chromosome 15, homologous with a human genome region (8q24) linked to bipolar disorder. Integrating the syntenic mouse QTL-interval with genotypes of 1868 BPD cases versus 14,311 control subjects revealed two associated genes (ADCY8 and KCNQ3). Adenylyl cyclase 8 (Adcy8) was differentially expressed in specific brain regions of mouse strains that differ in avoidance behavior levels. Finally, we showed that chronic infusion of the human mood stabilizer carbamazepine (that acts via adenylyl cyclase activity) significantly reduced mouse avoidance behavior, providing a further link between human mood disorders and this mouse home cage behavior. CONCLUSIONS: Our data suggest that Adcy8 might encode a translational behavioral endophenotype of bipolar disorder.

[Frequency of drug resistant tuberculosis in Poland in 2000 as compared to 1997]. / Czestosc wystepowania gruzlicy lekoopornej w Polsce w 2000 R. w porównaniu z 1997 r.

Prevalence of drug resistance to one drug and multidrug resistance--MDR in different categories of tuberculosis patients is an important information about the susceptibility pattern of Mycobacterium tuberculosis isolates against antimycobacterial drugs. Poland joined WHO/IUATLD global project on TB drug resistance surveillance, and carried out in 1996/1997 the first prospective survey, simultaneously on primary and acquired drug resistance. This study is repeated in 2000 according to the WHO/IUATLD protocol. The programme covered the whole country. A total of 16 regional centers participated in the co-operative study. 3705 questionnaires and cultures were obtained from patients who excreted TB bacilli during the 12-months from 1 st. January to 31st December 2000. Drug resistance tests to INH, RMP, SM, EMB were performed on Lowenstein-Jensen medium according to the proportion method or/and Bactec 460 TB system. 3705 TB patients (3037 new and 668 treated cases) bacteriologically confirmed by culture were included in one-year study. Primary resistance to any drug was found in 6.12% (CI 5.27-6.56) of new cases. 35 patients (1.15%, CI 0.77-1.35) were infected with MDR strains. Acquired resistance to any drug was found in 16.6% (CI 5.27-6.56), 8.53% (CL 6.41-9.6) of the patients who excreted MDR strains. We have found increased resistance from 3.6% in 1997 to 6.12% (p < 0.001) in 2000 and MDR from 0.6% in 1997 to 1.15% (p < 0.001) in 2000 in untreated tuberculosis patients in Poland. The rate of resistance in the group of treated TB patients was very similar in 1997 (17.0%) and in 2000 (16.6%); except 20% increase of MDR cases--(7.0% in 1997, and 8.53% in 2000). We observed an increase in drug resistant tuberculosis first time during 40 years long period of its monitoring. Regular monitoring of drug resistance in TB patients in Poland is recommended.

[Sensitivity of microscopy for detection of mycobacterium tuberculosis and MOTT (mycobacteria other than tuberculosis) on the basis of analysis 22.218 clinical materials submitted in 1998-2001 to the Department of Microbiology in National Tuberculosis and Lung Diseases Research Institute in Warsaw, Poland]. / Czulosc metody bakterioskopowej w wykrywaniu pratków gruzlicy i MOTT na podstawie analizy 22.218 badan diagnostycznych przeprowadzonych w Zakladzie Mikrobiologii IGiCHP w okresie 1998-2001.

The results of 22.218 respiratory specimens sent to our laboratory were studied to determine the sensitivity of the Ziehl-Neelsen (Z-N) stain and microscopy-fluorescence method for detection of M.tbc and MOTT. There were no AIDS patients among analyzed cases. Smears were positive for acid fast bacilli (AFB) 60.0% (480 of 800) of specimens growing M. tuberculosis and 25.1% (219 of 872) of specimens growing the six common species of MOTT. Smear positivity by species was 28.1% (141 of 502) for M. kansasii, 29.4% (43 of 146) for MAIC, 28.7% (35 of 122) for M. xenopi. No smear was positive for M. gordone (43 cultures), M. fortuitum (33 cultures), M. scrofulaceum (26 cultures). The rate of nonculturable mycobacteria on L-J medium was 0.2%; mean rate of contamination was 4.6%. We also analyzed the relation between the number of AFB seen on the smear and time of the growth of M. tuberculosis and relation between abundance of the culture growth and AFB seen on smears. These study suggest that the sensitivity of microscopy for Mycobacterium tuberculosis is comparable with the data of others authors. Sensitivity of microscopy is lower in MOTT detection than for M.tbc (p < 0.001). Time of growth and abundance of the M. tuberculosis cultures were adequate to AFB seen in microscopy.