LEARNING IMPULSE CONTROL IN A NOVEL ANIMAL MODEL: SYNAPTIC, CELLULAR, AND PHARMACOLOGICAL SUBSTRATES

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Mouse Uterine Natural Killer Cell Functions During Early Pregnancy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction.

Comparative seasonal acclimatization of food and energy consumption in adjacent populations of common spiny mice (Acomys cahirinus)

Animals inhabiting environments with low productivity and food availability commonly have reduced energy demands and increased digestive efficiencies. The dry matter intake (DMI), apparent digestible dry matter (ADDM), digestible efficiency (DE) and digestible energy intake (DEI) of two populations of common spiny mouse Acomys cahirinus were compared during both winter and summer under conditions of simulated water stress. Mice were captured from the north- and south-facing slopes (NFS and SFS) of the same canyon that represent mesic and xeric habitats, respectively. Measured variables were also compared between F-1 mice that had been born to either NFS or SFS mice, and raised in the laboratory. SFS mice were able to assimilate energy more efficiently than NFS mice during the summer. By comparison, NFS mice were able to assimilate more energy during the winter. During winter, NFS mice assimilated more energy at low levels of water stress, whereas SFS mice assimilated more energy at higher levels. Differences were also apparent in F-1 mice. It is therefore suggested that local climatic conditions can impose physiological adaptations that are retained in succeeding generations, creating unique meta-populations.

Comparative non-shivering thermogenesis in adjacent populations of the common spiny mouse (Acomys cahirinus) from opposite slopes: the effects of increasing salinity

We compared non-shivering thermogenesis between two adjacent populations of the common spiny mouse Acomys cahirinus from different habitats, in relation to increasing salinity. Individuals were captured from the north- and south-facing slopes of the same valley, that represent

Spatial genetic structuring in a vagile species, the European wood mouse

We examined the genetic structure of natural populations of the European wood mouse Apodemus sylvaticus at the microgeographic ( 30 km) scales. Ecological and behavioural studies indicate that this species exhibits considerable dispersal relative to its home-range size. Thus, there is potential for high gene flow over larger geographic areas. As levels of population genetic structure are related to gene flow, we hypothesized that population genetic structuring at the microgeographic level should be negligible, increasing only with geographic distance. To test this, four sites were sampled within a microgeographic scale with two additional samples at the macrogeographic level. Individuals (n=415) were screened and analysed for seven polymorphic microsatellite loci. Contrary to our hypothesis, significant levels of population structuring were detected at both scales. Comparing genetic differentiation with geographic distance suggests increasing genetic isolation with distance. However, this distance effect was non-significant being confounded by surprisingly high levels of differentiation among microgeographic samples. We attribute this pattern of genetic differentiation to the effect of habitat fragmentation, splitting large populations into components with small effective population sizes resulting in enhanced genetic drift. Our results indicate that it is incorrect to assume genetic homogeneity among populations even where there is no evidence of physical barriers and dispersal can occur freely. In the case of A. sylvaticus, it is not clear whether dispersal does not occur across habitat barriers or behavioural dispersal occurs without consequent gene flow.

Intraguild predation may explain an amphipod replacement: evidence from laboratory populations

In a laboratory experiment that permitted both observations of the behaviour of individuals and the monitoring of small populations, the role of 'intraguild predation' in the elimination of the freshwater amphipod Gammarus duebeni celticus by the introduced G. pulex was examined. Over 18 weeks, deaths in single and mixed species replicates were monitored. Rates of 'mortality' (deaths not due to cannibalism or predation) did not differ between the species. Gammarus cl. celticus, however, was more cannibalistic than G. pulex and, in both species, males were more often cannibalized than females. In mixed species replicates, the mean proportions of animals preyed upon did not differ among replicates with differing starting proportions of the two species, nor was there a difference between the sexes in numbers preyed upon. G. pulex, however, preyed more frequently on G. d celticus than vice versa, and this became more pronounced over time. In 87% of mixed species replicates, G. pulex eliminated G. d. celticus. The results support the proposition that intraguild predation may be the primary mechanism whereby G. pulex rapidly replaces G. d. celticus in freshwater. Integrating behavioural observations with population level monitoring may thus link pattern and process in behaviour and ecology.

Development and Single-Laboratory Validation of a Pseudofunctional Biosensor Immunoassay for the Detection of the Okadaic Acid Group of Toxins

A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 mu g/kg, a limit of detection of 31 mu g/kg, and a working range of 31-174 mu g/kg. The midpoint on the standard matrix calibration curve is 80 mu g/kg, half the current regulatory limit. Inter- and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were obtained using a certified reference material (R-2 = 0.99), laboratory reference material, and naturally contaminated mussel samples (R-2 = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins.

Single Laboratory Validation of a Surface Plasmon Resonance Biosensor Screening method for Paralytic Shellfish Poisoning Toxins

A research element of the European Union (EU) sixth Framework project BioCop focused on the development of a surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins in shellfish as an alternative to the increasingly ethically unacceptable mouse bioassay. A biosensor assay was developed using both a saxitoxin binding protein and chip surface in tandem with a highly efficient simple extraction procedure. The present report describes the single laboratory validation of this immunological screening method, for this complex group of toxins with differing toxicities, according to the European Decision 2002/657/EC in conjunction with IUPAC and AOAC single laboratory validation guidelines. The different performance characteristics (detection capability CC beta, specificity/selectivity, repeatability, reproducibility, stability, and applicability) were determined in relation to the EU regulatory limit of 800 mu g of saxitoxin equivalents (STX eq) per kg of shellfish meat. The detection capability CC beta was calculated to be 120 mu g/kg. Intra-assay repeatability was found to be between 2.5 and 12.3% and interassay reproducibility was between 6.1 and 15.2% for different shellfish matrices. Natural samples were also evaluated and the resultant data displayed overall agreements of 96 and 92% with that of the existing AOAC approved methods of mouse bioassay (MBA) and high performance liquid chromatography (HPLC), respectively.

Myocardial infarction in the C57BL/6J mouse - A quantifiable and highly reproducible experimental model

Introduction: The laboratory mouse is a powerful tool in cardiovascular research. In this report, we describe a method for a reproducible mouse myocardial infarction model that would allow subsequent comparative and quantitative studies on molecular and pathophysiological variables. Methods: (A) The distribution of the major coronary arteries including the septal artery in the left ventricle of the C57BL/6J mice (n=20) was mapped by perfusion of latex dye or fluorescent beads through the aorta. (B) The territory of myocardial infarction after the ligation of the most proximal aspect of the left anterior descending (LAD) coronary artery was quantified. (C) The consistency in the histological changes parallel to the infarction at different time points was analyzed. Results: (A) The coronary artery tree of the mouse is different from human and, particularly, in regard to the blood supply of the septum. (B) Contrary to previous belief, the septal coronary artery in the mouse is variable in origin. (C) A constant ligation of the LAD immediately below the left auricular level ensures a statistically significant reproducible infarct size. (D) The ischemic changes can be monitored at a histological level in a way similar to what is described in the human. Conclusion: We illustrate a method for maximal reproducibility of experimental acute myocardial infarction in the mouse model, due to a consistent loss of perfusion in the lower half of the left ventricle. This will allow the study of molecular and physiological variables in a controlled and quantifiable experimental model environment. (C) 2004 Elsevier Inc. All rights reserved.

Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated that allows for the simultaneous detection of medroxyprogesterone acetate (MPA), megestrol acetate (MGA), melengestrol acetate (MLA), chlormadinone acetate (CMA) and delmadinone acetate (DMA) in animal kidney fat using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds were extracted from kidney fat using acetonitrile, defatted using a hexane wash and subsequent saponification. Extracts were then purified on Isolute CN solid-phase extraction cartridges and analysed by LC-MS/MS. The method was validated in animal kidney fat in accordance with the criteria defined in Commission Decision 2002/657/EC. The decision limit (CC) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 g kg-1, respectively, for MPA, MGA, MLA, DMA and CMA, with respective detection capability (CC) values of 0.20, 0.81, 0.68, 1.07 and 0.92 g kg-1. The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26% for MPA, MGA, MLA, DMA and CMA, respectively. Fortifying kidney fat samples (n = 18) in three separate assays showed the accuracy of the method to be between 98 and 100%. The precision of the method, expressed as % RSD, for within-laboratory reproducibility at three levels of fortification (1, 1.5 and 2 g kg-1 for MPA, 5, 7.5 and 10 g kg-1 for MGA, MLA, DMA and CMA) was less than 5% for all analytes.

EFFECTIVE LABORATORY MONITORING FOR THE ABUSE OF THE BETA-AGONIST CLENBUTEROL IN CATTLE

The use of the beta-agonist clenbuterol (CBL) as a growth promoter has been outlawed in European meat production. The detection of its illegal use is dependent on CBL residues persisting in animal tissues for longer than the withdrawal times given by abusers. A comparison of urine, bile and liver matrices indicated that analysis of the liver offered the best possibility for CBL detection. However, an experimental study showed that CBL detection following withdrawal could be further extended (up to 56 d) if the retina was used as the target tissue. Analysis of 703 retina and liver samples from cattle suspected of CBL medication revealed that 96 cattle had CBL residues present in their retinas, only 46 of these were liver positive. There were no instances of liver CBL residues being detected without the associated retina also being positive.

POPULATION BIOLOGY OF HELIGMOSOMOIDES-POLYGYRUS (NEMATODA) IN THE WOOD MOUSE

1. This paper examines the interaction between the wood mouse, Apodemus sylvaticus L., and the intestinal nematode, Heligmosomoides polygyrus Dujardin, using data collected at Tollymore Park Forest, Co. Down, Northern Ireland, between November 1978 and July 1981.

DISPERSION IN THE WOOD MOUSE, APODEMUS-SYLVATICUS - VARIABLE RESOURCES IN TIME AND SPACE

(1) The abundance and dispersion of a population of Apodemus sylvaticus was investigated with respect to tree seed availability and vegetative structure over three harvests.