This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

Methylmercury (MeHg) is an environmental toxin to which we are exposed through the consumption of seafood. Reactive oxygen species (ROS) have been implicated in the mechanism of toxicity, and in vitro studies in our laboratory have implicated DNA oxidation, particularly the DNA repair enzyme oxoguanine glycosylase 1 (OGG1). My studies determined the effects of in utero exposure to MeHg on fetal brain DNA oxidation and postnatal neurodevelopmental deficits, and the role of ROS-mediated oxidative DNA damage using the free radical spin trap, α-phenyl-N-tert-butylnitrone (PBN), and DNA repair-deficient ogg1 knockout mice. While neither MeHg nor PBN altered DNA oxidation in fetal brain, MeHg caused cognitive deficits in passive avoidance and novel object recognition, the latter of which was blocked by PBN pretreatment, suggesting ROS involvement. Preliminary longevity studies following one litter from each treatment group to 16 months suggest that in utero MeHg treatment may shorten lifespan. Endogenous DNA oxidation was increased in the brains of ogg1 knockout fetuses compared to wild-type littermates, although this was not enhanced by MeHg. However, OGG1-deficient animals exhibited cognitive deficits in passive avoidance after MeHg treatment, suggesting a role for DNA damage. Furthermore, ogg1 knockout female mice exhibited a passive avoidance deficit compared to wild-type females regardless of treatment, corroborating a role for oxidative DNA damage in neurodevelopmental deficits. MeHg increased apoptosis in the hippocampal region of fetal brain, and may cause DNA double-strand breaks (DSBs), evidenced by enhanced phosphorylation of histone 2AX (γH2AX). Ogg1 knockout progeny exhibited increased cellular proliferation or migration in the developing hippocampal region, which was blocked by MeHg. My results provide the first evidence that: (1) MeHg may decrease lifespan; (2) PBN protects against some postnatal neurodevelopmental deficits caused by in utero exposure to MeHg; and (3) DNA repair-deficient progeny are more susceptible to postnatal cognitive deficits caused by in utero MeHg exposure, suggesting that ROS-mediated DNA oxidation plays a role in MeHg-initiated neurodevelopmental deficits.

Dopamine D3 receptors (DRD3) are implicated in relapse to drugs. The current study investigated the role of DRD3 in cue-induced reinstatement of nicotine-seeking in rats. Rats were trained to lever-press for intravenous infusions of nicotine, associated with the illumination of a cue-light, under a fixed-ratio schedule of reinforcement. Following extinction of the behaviour, where lever pressing had no consequences, reinstatement testing was performed by reintroduction of the cues after systemic or local administration (into discrete brain areas) of the DRD3 selective antagonist SB277011-A. Systemic antagonism of DRD3 significantly attenuated cue-induced reinstatement of nicotine-seeking. The same effect was observed upon infusions of SB277011-A into the basolateral amygdala or the lateral habenula, but not the nucleus accumbens. The current findings implicate DRD3 in cue-induced reinstatement of nicotine, delineate some of the neural substrates underlying this role and support a potential for using selective DRD3 antagonists for the prevention of relapse to smoking.

Pathological Gambling (PG) is an impulse control disorder with lifetime prevalence of 1-3%. Available treatments are limited by uncertain classification and complexity of implicated neurotransmitter systems. Dopamine (DA), a key neurotransmitter implicated in addictive behavior and reward is elevated in response to gambling and psychostimulants. Based on previous research, it was hypothesized that the D2 blocker, haloperidol (HAL), will enhance slot machine reinforcement in PG but not in Healthy Controls (HC). If this increase reflects preferential stimulation of D1 receptors and group differences in D1 sensitivity, D1-D2 blocker (fluphenazine, FLU) should offset increase in reinforcement seen with HAL in PG subjects. In line with DA's implicated role in 'wanting' vs. 'liking' of the addictive reinforcer, the results suggest that DA release mediated partial D1 activation under FLU led to clear differentiation between groups with increased 'wanting' seen in controls but not in gamblers. DA's role in 'liking' however remains elusive.

Dopamine D3 receptors (DRD3) are implicated in relapse to drugs. The current study investigated the role of DRD3 in cue-induced reinstatement of nicotine-seeking in rats. Rats were trained to lever-press for intravenous infusions of nicotine, associated with the illumination of a cue-light, under a fixed-ratio schedule of reinforcement. Following extinction of the behaviour, where lever pressing had no consequences, reinstatement testing was performed by reintroduction of the cues after systemic or local administration (into discrete brain areas) of the DRD3 selective antagonist SB277011-A. Systemic antagonism of DRD3 significantly attenuated cue-induced reinstatement of nicotine-seeking. The same effect was observed upon infusions of SB277011-A into the basolateral amygdala or the lateral habenula, but not the nucleus accumbens. The current findings implicate DRD3 in cue-induced reinstatement of nicotine, delineate some of the neural substrates underlying this role and support a potential for using selective DRD3 antagonists for the prevention of relapse to smoking.

Pathological Gambling (PG) is an impulse control disorder with lifetime prevalence of 1-3%. Available treatments are limited by uncertain classification and complexity of implicated neurotransmitter systems. Dopamine (DA), a key neurotransmitter implicated in addictive behavior and reward is elevated in response to gambling and psychostimulants. Based on previous research, it was hypothesized that the D2 blocker, haloperidol (HAL), will enhance slot machine reinforcement in PG but not in Healthy Controls (HC). If this increase reflects preferential stimulation of D1 receptors and group differences in D1 sensitivity, D1-D2 blocker (fluphenazine, FLU) should offset increase in reinforcement seen with HAL in PG subjects. In line with DA's implicated role in 'wanting' vs. 'liking' of the addictive reinforcer, the results suggest that DA release mediated partial D1 activation under FLU led to clear differentiation between groups with increased 'wanting' seen in controls but not in gamblers. DA's role in 'liking' however remains elusive.

Rationale and objectives: Alcohol and nicotine are the most commonly abused drugs and they are often taken together. To help address some of clinical issues regarding nicotine and alcohol co-dependence, a procedure in which rats self-administer nicotine intravenously and alcohol orally during the same operant session has been developed. Methods: Male Wistar rats were trained to self-administer alcohol (12%, w/v; 0.19 ml/delivery) or implanted with jugular catheters and trained to self-administer nicotine (30 μg/kg IV/infusion) by pressing a lever or were trained to self-administer both drugs, some with alcohol first, and others with nicotine first. Results: Animals readily coadministered alcohol and nicotine concurrently. Access to alcohol reduced nicotine selfadministration significantly. Conclusions: These results show that rats will self-administer relevant amounts of intravenous nicotine and oral alcohol concurrently. They also provide further support for the important relationship between nicotine and alcohol.

The underlying mechanism of morphine tolerance remains unclear. EphB2 regulates synaptic efficiency with respect to learning and memory. Previously, we demonstrated that loss of EphB2 significantly accelerates the rate of morphine tolerance and alters behavioural responses to morphine following tolerance. However, EphB2 null mice exhibit no significant alteration in their metabolism of morphine compared to littermate controls, or altered mu opioid receptor expression levels within the spinal cord or brain compared to littermate controls. Therefore, we investigated whether loss of EphB2 alters learned responsiveness to morphine through modification of hippocampal function. Interestingly, results indicate that electrolytic lesions of the dorsal hippocampus of wild-type mice display similar behavioural responses seen in EphB2 null mice compared to sham operated controls. These findings suggest that loss of EphB2 function within the hippocampus is a critical feature in mediating morphine-dependent tolerance, and suggests a novel role for EphB2 receptor signaling in opiate-dependent learning.

Recent studies have demonstrated that an acute, low dose of ketamine, a non-competitive NMDA receptor antagonist, provides rapid and sustained antidepressant effects in patients with major depressive disorder. Studies in rodents have shown that the antidepressant properties of ketamine are due to an increase in dendritic spine density in the cortex. Our goal was to determine whether these effects are specific to ketamine and whether they are dependent on dose, drug regimen and brain region. We observed that the effects of ketamine on spine density were dependent on dose and drug regimen and were also brain region specific. In addition, MK-801, another NMDA receptor antagonist, did not demonstrate the same effects on spine density as ketamine. Furthermore, genetic NMDA receptor hypofunction significantly reduced spine density. Our studies demonstrate that while acute ketamine treatment leads to an increase in cortical spine density, chronic administration has opposite and potentially detrimental effects.