In a honey bee colony, the queen uses queen mandibular pheromone (QMP) to induce young worker bees to feed and groom her. Among its many behavioural and physiological effects, QMP reduces dopamine levels in the brains of young worker bees. Dopamine is a biogenic monoamine involved in numerous functions including motor control and aversive learning. This study investigates the effects of QMP on motor activities and aversive learning behaviour and the potential link between QMP and dopamine levels in the brain of young bees. In young bees under the age of 15-days, QMP dramatically reduced locomotor activity and inhibited aversive learning behaviour. Interestingly in older bees these behaviours were not affected by pheromone. Treating young bees with the dopamine precursor, L-dopa (3.25 [mu]g/mI), partially rescued the levels of locomotor activity in QMP-treated bees, and reduced QMP�s effects on aversive learning. This suggests that blocking effects of QMP on both locomotor activity and aversive learning result at least in part from QMP-induced changes in brain dopamine levels. Two components of the QMP blend, 4-hydroxy-3-methoxyphenylethanol (HVA) and methyl p-hydroxybenzoate (HOB) were examined more closely. Both HVA and HOB are structurally similar to dopamine. HVA was found to mimic the effects of the full QMP blend on aversive learning. Treating bees with HVA reduced aversive learning in young bees. In contrast, treatment with HOB did not affect learning ability. This strongly suggests that HVA is one of the key components that mediates the actions of QMP on aversive learning. The final section of this thesis investigates why it might be advantageous to honey bee queens to block aversive learning and reduce locomotor activity in young worker bees. The study reveals age-related differences in behaviours that individual worker bees display towards QMP. Young bees reared with QMP or collected from a queenright hive showed attraction to QMP. Conversely, older bees displayed avoidance behaviour towards QMP. By blocking the establishment of aversive memories, young bees may be prevented from forming an association between QMP and any unpleasant side effects induced by this pheromone. This may confer significant benefit to the queen by increasing the likelihood of young workers remaining in her attendance.

Dopamine appears to play a critical role in regulating spatial working memory (SWM) in non-human primates, and SWM deficits are observed in patients with Parkinson�s disease and schizophrenia. Unfortunately, the current experimental literature in humans is marred by inconsistent behavioural findings, and there is a void in neuroimaging studies examining dopaminergic manipulation of SWM-related brain activity. The present thesis used a combination of behavioural neurocognitive testing and brain imaging to further examine dopaminergic manipulation of SWM in healthy humans, using two pharmacological challenges: 1) acute tyrosine depletion (TPD) (to acutely deplete tonic dopamine), and 2) D1/D2 receptor activation using the dopamine receptor agonist pergolide (to stimulate dopamine neurotransmission) under conditions of TPD. The effects of TPD on behavioural performance were examined using three SWM tasks: 1) a delayed-recognition task previously impaired by TPD (Experiment 1) and 2) two delayed-response tasks designed to vary only in response requirements (Experiment 2). The findings demonstrated an apparent failure of TPD to impair performance on any of the tasks. Further, the effects of TPD on SWM-related brain activity during a SWM n-back task were examined using regional Cerebral Blood Flow (rCBF) measured by H2 150 Positron Emission Tomography (Experiment 2), and Steady State Visually Evoked Potentials (SSVEP) measured by Steady State Probe Topography (Experiment 4). TPD failed to produce discernable effects on either neural networks (task-related rCBF) or temporal electrophysiological activity (SSVEP) associated with the SWM n-back task. In contrast, D1/D2 receptor stimulation under dopamine depleted conditions impaired performance on both a SWM delayed-response task (Experiment 1) and SWM n-back task (Experiment 2), and resulted in task-related increases in fronto-temporal SSVEP latency (suggestive of increased inhibition) and decreases in parieto-occipital SSVEP amplitude (suggestive of increased activation) during the early delay period of the SWM n-back task (Experiment 4). These changes are consistent with the undisputed role of frontal and parietal regions in n-back task performance, and with previous evidence of dopaminergic modulation of these regions in animals and humans. In summary, TPD did not modulate SWM behavioural performance on four different SWM tasks with differing task demands, and failed to produce measurable changes to either SWM-related neural networks (task-related rCBF) or cortical electrophysiological activity (SSVEP) associated with the SWM n-back task. The implication of these findings, when taken together with previous studies, is that the degree of dopaminergic depletion achieved with TPD may be insufficient to consistently and robustly modulate SWM networks in healthy humans, questioning the utility of TPD as a probe of dopaminergic function. In addition, these findings demonstrate the complexity of stimulating D1/D2 receptors under dopamine depleted conditions, and highlight the critical importance of baseline dopamine levels in influencing the effects of acute dopamine challenge on SWM performance.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Recent research has found that perceptual deficits exist in Parkinson’s disease (PD), yet the link between perception and movement impairments is not well understood. Inaccurate estimation of distance has the potential to be an underlying cause of movement impairments. Alternatively, those with PD may not be able to perceive their own movements accurately. The main objective of this thesis was to evaluate (1) whether distance estimation is influenced by static perception compared to perception during movement in PD, (2) how visual motion processing contributes to distance estimation during movement, and (3) how dopaminergic medication contributes to these distance estimation deficits. Thirty-seven participants (19 individuals with PD, 18 age-matched healthy control participants (HC) estimated distance to a remembered target in a total of 48 trials, in 4 randomized blocks. Estimation conditions included: (i) no motion: participants pointed with a laser, (ii) motion: participants walked to the estimated position, (iii) visual motion (wheelchair): participants were pushed in a wheelchair while they gave their estimate, (iv) visual motion (VR): participants completed their distance estimate while seated and viewed themselves (as if they were walking) in VR. PD patients completed this protocol twice; once OFF and once ON dopaminergic medication. Participants were matched for age, distance acuity, Modified Mini Mental State Exam (3MS), spatial working memory and motor planning ability. In Study 1 (no motion vs. motion), individuals with PD and healthy control participants did not differ in judgment accuracy during the no motion condition. However, those with PD did have greater amounts of error compared to healthy control participants while estimating distance during the motion condition. Similarly, those with PD significantly underestimated the target position compared to healthy control participants during the motion condition only. Individuals with PD demonstrated greater variability overall. In Study 2, error did not differ between PD and HC groups during visual motion perception (wheelchair). Interestingly, the HC group tended to perform significantly worse than those with PD in the VR condition. Overall, across both studies there was no significant influence of dopaminergic medication in any of the conditions. Individuals with PD demonstrated distance estimation deficits only when required to move through their environment. In contrast to estimations made with movement, neither static estimation nor estimations made with visual motion revealed significant differences between the two groups. Thus perceptual estimation deficits appear to occur only during movement, which may be suggestive of an underlying sensory processing deficit which leads to a problem integrating vision and self-motion information.

No description available.

No description available.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

The aim of this thesis was the functional characterization of the zebrafish parl (Presenilin-Associated Rhomboid-Like) genes which code for mitochondrial proteins involved in cell survival. A mutation in PARL has been described in Parkinson’s disease patients. I investigated the role of mitochondrial PD-related proteins using a zebrafish parla and parlb deficiency model. I found that the knockdown of both parl genes is lethal. Parla plays a larger role in patterning of the DA neurons in the ventral diencephalon than Parlb. The human PARL rescued the double morphant phenotype, suggesting function conservation between zebrafish and humans. I was able to rescue the mortality and DA neuron mispatterning observed in double morphants with synthetic pink1 mRNA. This suggests that parl genes are epistatic to pink1 in zebrafish. To visualize mitochondria specifically in dopaminergic neurons of live zebrafish, I established a transgenic line Tg(dat:tom20 MLS-mCherry) where regulatory elements of the dopamine transporter (dat) were used to drive expression of a Tom20-mCherry fusion protein that is targeted to the mitochondria. I characterised the expression of Tom20-mCherry to the mitochondria of the majority of DA neuron groups. In addition, I observed a decrease in mCherry fluorescence following MPTP exposure of live fish. The PD-related mutation in PARL is located in a cleavage site of the mammalian protein, which is necessary for the production of the beta peptide; however, this site is predicted to be absent in the zebrafish Parls. To establish the cleavage patterns of the zebrafish Parls and compare them to those of human PARL, I examined the cleavage of Parl-Flag constructs in cultured cells. I detected one band for Parla-Flag and two bands representing Parlb-Flag. The parla and parlb deficiency model along with the characterization of the cleavage patterns of Parl and the Tg(dat:tom20 MLS-mCherry) transgenic line are tools which will help elucidate the role of mitochondrial proteins in PD research.