Case studies : Using the zebrafish to evaluate neurobehavioral phenotypes

The use of zebrafish in behavioral neuroscience is rapidly growing. Zebrafish can be assessed for alterations in multiple behavioral endpoints, creating opportunities to use this powerful model to identify chemicals that alter behavioral phenotypes. To evaluate the utility of zebrafish for neurotoxicity research, we designed custom instrumentation to evaluate numerous embryonic and adult zebrafish behaviors. PRAT or Photomotor Response Analysis Tool was used to analyze the embryonic photomotor response (EPR) behavior in embryonic zebrafish (24 hours post fertilization). Shuttleboxes were used to evaluate learning and active avoidance conditioning and a zebrafish Visual Imaging System (zVIS) was used to measure fear responses. Social behavior was observed using Viewpoint tracking software. Startle responses were also analyzed using taps and Noldus Ethiovision XT tracking software. EPR results showed differential movement activities throughout development of larval zebrafish. Highest movement peaks were seen in 35-37 hours post fertilization fish. Using these custom analysis tools, we also evaluated the impact of Vitamin E deficiency and developmental Benzo[a]pyrene exposure on complex adult behaviors. Generational effects of BaP exposure were also tested. Zebrafish were fed defined-diets that either had sufficient or deficient levels of Vitamin E. The vitamin E deficient zebrafish had a ~30% decrease in learning rate relative to the fish with sufficient levels of Vitamin E. Startle response data showed that vitamin E deficient fish do not get desensitized to tap stimulus. Three exposure groups and generations were reared and spawned for the BaP study (0.1% DMSO controls, 1.25 ppm BaP, 2.5 ppm BaP). The zVIS system consists of an array of 8 tanks with only single side views of video projections on LCD monitors. This allows individual fish to visualize either a group of swimming zebrafish or single predator fish. For the socialization assay zebrafish were tracked using Viewpoint tracking software. Distances apart from each other were measured and analyzed in BaP exposed fish. For the predator test, zebrafish were expected to move away from the screen. The proximity of the zebrafish is tracked relative to the LCD screen projections. The preliminary results from BaP exposed zebrafish and 0.1% DMSO controls showed the percent of time spent away from the screen during the predator test or fear response assay was in the high 70% range for all fish. The 2.5 ppm BaP fish had on average the highest percentage (65% vs 50%) time spent away from the screen. Although it is uncertain as of now if there are any generational effects because further analysis is needed. Preliminary shoaling data shows that shoaling speed may be affected by DMSO exposure. The use of DMSO controls may not be optimal for this study. Disassociation is seen in both 1.25 ppm BaP and 2.5 ppm BaP exposure groups in the F2 generation. Collectively, these data demonstrate that custom behavioral systems are able to measure complex behavioral phenotypes and suggests that there are enormous opportunities for translation neurotoxicity research using zebrafish

Improved X-ray detection and particle identification with avalanche photodiodes

Avalanche photodiodes are commonly used as detectors for low energy x-rays. In this work we report on a fitting technique used to account for different detector responses resulting from photo absorption in the various APD layers. The use of this technique results in an improvement of the energy resolution at 8.2 keV by up to a factor of 2, and corrects the timing information by up to 25 ns to account for space dependent electron drift time. In addition, this waveform analysis is used for particle identification, e.g. to distinguish between x-rays and MeV electrons in our experiment.Comment: 6 pages, 6 figure

Measurement of Upsilon production in pp collisions at \sqrt{s} = 7 TeV

The production of Upsilon(1S), Upsilon(2S) and Upsilon(3S) mesons in proton-proton collisions at the centre-of-mass energy of sqrt(s)=7 TeV is studied with the LHCb detector. The analysis is based on a data sample of 25 pb-1 collected at the Large Hadron Collider. The Upsilon mesons are reconstructed in the decay mode Upsilon -> mu+ mu- and the signal yields are extracted from a fit to the mu+ mu- invariant mass distributions. The differential production cross-sections times dimuon branching fractions are measured as a function of the Upsilon transverse momentum pT and rapidity y, over the range pT < 15 GeV/c and 2.0 < y < 4.5. The cross-sections times branching fractions, integrated over these kinematic ranges, are measured to be sigma(pp -> Upsilon(1S) X) x B(Upsilon(1S)->mu+ mu-) = 2.29 {\pm} 0.01 {\pm} 0.10 -0.37 +0.19 nb, sigma(pp -> Upsilon(2S) X) x B(Upsilon(2S)->mu+ mu-) = 0.562 {\pm} 0.007 {\pm} 0.023 -0.092 +0.048 nb, sigma(pp -> Upsilon(3S) X) x B(Upsilon(3S)->mu+ mu-) = 0.283 {\pm} 0.005 {\pm} 0.012 -0.048 +0.025 nb, where the first uncertainty is statistical, the second systematic and the third is due to the unknown polarisation of the three Upsilon states.Comment: 22 pages, 7 figure

Garcia_BRR_Thesis.pdf

The use of zebrafish in behavioral neuroscience is rapidly growing. Zebrafish can be assessed for alterations in multiple behavioral endpoints, creating opportunities to use this powerful model to identify chemicals that alter behavioral phenotypes. To evaluate the utility of zebrafish for neurotoxicity research, we designed custom instrumentation to evaluate numerous embryonic and adult zebrafish behaviors. PRAT or Photomotor Response Analysis Tool was used to analyze the embryonic photomotor response (EPR) behavior in embryonic zebrafish (24 hours post fertilization). Shuttleboxes were used to evaluate learning and active avoidance conditioning and a zebrafish Visual Imaging System (zVIS) was used to measure fear responses. Social behavior was observed using Viewpoint tracking software. Startle responses were also analyzed using taps and Noldus Ethiovision XT tracking software. EPR results showed differential movement activities throughout development of larval zebrafish. Highest movement peaks were seen in 35-37 hours post fertilization fish. Using these custom analysis tools, we also evaluated the impact of Vitamin E deficiency and developmental Benzo[a]pyrene exposure on complex adult behaviors. Generational effects of BaP exposure were also tested. Zebrafish were fed defined-diets that either had sufficient or deficient levels of Vitamin E. The vitamin E deficient zebrafish had a ~30% decrease in learning rate relative to the fish with sufficient levels of Vitamin E. Startle response data showed that vitamin E deficient fish do not get desensitized to tap stimulus. Three exposure groups and generations were reared and spawned for the BaP study (0.1% DMSO controls, 1.25 ppm BaP, 2.5 ppm BaP). The zVIS system consists of an array of 8 tanks with only single side views of video projections on LCD monitors. This allows individual fish to visualize either a group of swimming zebrafish or single predator fish. For the socialization assay zebrafish were tracked using Viewpoint tracking software. Distances apart from each other were measured and analyzed in BaP exposed fish. For the predator test, zebrafish were expected to move away from the screen. The proximity of the zebrafish is tracked relative to the LCD screen projections. The preliminary results from BaP exposed zebrafish and 0.1% DMSO controls showed the percent of time spent away from the screen during the predator test or fear response assay was in the high 70% range for all fish. The 2.5 ppm BaP fish had on average the highest percentage (65% vs 50%) time spent away from the screen. Although it is uncertain as of now if there are any generational effects because further analysis is needed. Preliminary shoaling data shows that shoaling speed may be affected by DMSO exposure. The use of DMSO controls may not be optimal for this study. Disassociation is seen in both 1.25 ppm BaP and 2.5 ppm BaP exposure groups in the F2 generation. Collectively, these data demonstrate that custom behavioral systems are able to measure complex behavioral phenotypes and suggests that there are enormous opportunities for translation neurotoxicity research using zebrafish.Keywords: Zebrafish, Neurobehaviora

Garcia_BRR_Seminar.pdf

The use of zebrafish in behavioral neuroscience is rapidly growing. Zebrafish can be assessed for alterations in multiple behavioral endpoints, creating opportunities to use this powerful model to identify chemicals that alter behavioral phenotypes. To evaluate the utility of zebrafish for neurotoxicity research, we designed custom instrumentation to evaluate numerous embryonic and adult zebrafish behaviors. PRAT or Photomotor Response Analysis Tool was used to analyze the embryonic photomotor response (EPR) behavior in embryonic zebrafish (24 hours post fertilization). Shuttleboxes were used to evaluate learning and active avoidance conditioning and a zebrafish Visual Imaging System (zVIS) was used to measure fear responses. Social behavior was observed using Viewpoint tracking software. Startle responses were also analyzed using taps and Noldus Ethiovision XT tracking software. EPR results showed differential movement activities throughout development of larval zebrafish. Highest movement peaks were seen in 35-37 hours post fertilization fish. Using these custom analysis tools, we also evaluated the impact of Vitamin E deficiency and developmental Benzo[a]pyrene exposure on complex adult behaviors. Generational effects of BaP exposure were also tested. Zebrafish were fed defined-diets that either had sufficient or deficient levels of Vitamin E. The vitamin E deficient zebrafish had a ~30% decrease in learning rate relative to the fish with sufficient levels of Vitamin E. Startle response data showed that vitamin E deficient fish do not get desensitized to tap stimulus. Three exposure groups and generations were reared and spawned for the BaP study (0.1% DMSO controls, 1.25 ppm BaP, 2.5 ppm BaP). The zVIS system consists of an array of 8 tanks with only single side views of video projections on LCD monitors. This allows individual fish to visualize either a group of swimming zebrafish or single predator fish. For the socialization assay zebrafish were tracked using Viewpoint tracking software. Distances apart from each other were measured and analyzed in BaP exposed fish. For the predator test, zebrafish were expected to move away from the screen. The proximity of the zebrafish is tracked relative to the LCD screen projections. The preliminary results from BaP exposed zebrafish and 0.1% DMSO controls showed the percent of time spent away from the screen during the predator test or fear response assay was in the high 70% range for all fish. The 2.5 ppm BaP fish had on average the highest percentage (65% vs 50%) time spent away from the screen. Although it is uncertain as of now if there are any generational effects because further analysis is needed. Preliminary shoaling data shows that shoaling speed may be affected by DMSO exposure. The use of DMSO controls may not be optimal for this study. Disassociation is seen in both 1.25 ppm BaP and 2.5 ppm BaP exposure groups in the F2 generation. Collectively, these data demonstrate that custom behavioral systems are able to measure complex behavioral phenotypes and suggests that there are enormous opportunities for translation neurotoxicity research using zebrafish.Keywords: Zebrafish, NeurobehavioralKeywords: Zebrafish, Neurobehaviora

Frequency of Th17 cells correlates with the presence of lung lesions in pigs chronically infected with Actinobacillus pleuropneumoniae

International audienceAbstractPorcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6–10 days post-infection (dpi)] or the chronic phase of APP infection (27–31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4+CD8αdim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4+ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4+CD8αdimIL-17A+) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection

DataSheet_1_Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.docx

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.</p

Phenotypic Characterization of a Virulent PRRSV-1 Isolate in a Reproductive Model With and Without Prior Heterologous Modified Live PRRSV-1 Vaccination

Reproductive disorders induced by porcine reproductive and respiratory syndrome virus (PRRSV) cause high economic losses in the pig industry worldwide. In this study, we aimed to phenotypically characterize a virulent PRRSV-1 subtype 1 isolate (AUT15-33) in a reproductive model. Furthermore, the protective effect of a heterologous modified live virus vaccine (ReproCyc® PRRS EU) was evaluated. In addition, PRRSV AUT15-33 was genotypically compared to other well-characterized isolates. Sixteen gilts were equally divided into four groups: a vaccinated and infected group (V-I), a vaccinated and non-infected group (V-NI), a non-vaccinated and infected group (NV-I), and a non-vaccinated and non-infected (NV-NI) group. After PRRSV infection on gestation day 84, all gilts were clinically examined on a daily basis, and blood samples were taken at five timepoints. Necropsy was performed 3 weeks after infection. The fetal preservation status was assessed, and PRRSV RNA concentrations were measured in the blood and tissue samples from all gilts and fetuses. After infection, all four gilts in the NV-I group were viremic throughout 17 days post-infection (dpi), whereas two gilts in the V-I group were viremic at only one timepoint at 6 dpi. The viral load was significantly higher in gilt serum, tracheobronchial lymph nodes, uterine lymph nodes, maternal endometrium, and fetal placenta of NV-I gilts compared to the V-I ones (p< 0.05). Moreover, the preservation status of the fetuses derived from NV-I gilts was significantly impaired (55.9% of viable fetuses) compared to the other groups (p< 0.001). Upon comparison with other known isolates, the phylogenetic analyses revealed the closest relation to a well-characterized PRRSV-1 subtype 1 field isolate from Belgium. In conclusion, the high virulence of AUT15-33 was phenotypically confirmed in an experimental reproductive model. The vaccination of the gilts showed promising results in reducing viremia, fetal damage, and transplacental transmission of the PRRSV-1 strain characterized in this study