In the peripheral nervous system (PNS), aging is associated with a number of disorders, including a decline in regenerative capacity after injury. Although this decline has been observed in both rodents and humans for decades, the cellular and molecular underpinnings of this defect have remained elusive. As such, the goal of this thesis was to elucidate, at least in part, how aging impinges on axonal regeneration.

Predictions help guide learning. As we encounter objects in our environment, we make predictions about their value. When outcomes match our predictions, learning is not required. When outcomes are unexpected, however, we update our predictions to reflect our experience. Dopamine neurons are thought to facilitate this process by encoding reward prediction error, or the difference between actual and predicted reward. Despite decades of work on prediction errors and their role in learning, little is known about how they are calculated in the brain. To determine how dopamine neurons calculate prediction error, I combined optogenetic manipulations with extracellular recordings while mice engaged in classical conditioning. In Chapter 1, I demonstrate that dopamine neurons perform subtraction, a computation that is ideal for reward learning but rarely observed in the brain. Furthermore, by carefully examining how individual dopamine neurons respond to various sizes of reward and levels of expectation, I reveal striking homogeneity from neuron to neuron. All dopamine neurons appear to follow the same function, just scaled up or down. This universal template ensures robust information coding, allowing each dopamine neuron to accurately calculate reward prediction error and broadcast this information to other brain areas vital for learning. In Chapter 2, I attempt to uncover the inputs that dopamine neurons use to calculate prediction errors. In particular, I test the hypothesis that a group of inhibitory neurons surrounding dopamine neurons in the midbrain may provide information about expected reward. By selectively exciting and inhibiting these nearby neurons, I discover that they indeed play a causal role in prediction errors, inhibiting dopamine neurons when reward is expected. Together, my results help uncover the arithmetic and local circuitry underlying dopamine prediction errors.

All animals must adapt their behaviors by experience to survive. In mammals, this adaptive process is thought occur through a synaptic loop involving the cortex, basal ganglia (BG) and thalamus. Here we use transgenic mice and novel recombinant viruses (Chapter 1) to explore the brain circuits that underlie this interaction. Our focus is on how cell types within the BG affect cortical feedback during development and in adulthood. Accepted models postulate that the BG modulate cerebral cortex 1) indirectly via an inhibitory output to thalamus and that this thalamic output is 2) bi-directionally controlled from within the BG by striatal direct (dSPNs) and indirect (iSPNs) pathway spiny neurons. In Chapter 2, we show that activity in iSPNs and dSPNs plays a complementary role in the post-natal synaptic wiring of the BG. Inhibiting iSPNs or dSPNs results in opposite changes in the number of excitatory synapses made onto SPNs from cortical and thalamic inputs. Our results suggest that the cortex-BG-thalamus function in a closed-loop and balanced iSPN/dSPN activity is required for proper synaptic wiring during development. In Chapter 3, we describe a non-thalamic output of the BG to the frontal cortex (FC) emanating from globus pallidus externus (GP). The GP-FC projection consists of two cell types that release GABA and GABA/Acetylcholine, mostly onto cortical interneurons, with the net effect of increasing cortical firing rate. These results suggest that iSPNs and dSPNs can affect cortical output through GP-based disinhibition in addition to thalamus-based excitation. Moreover, GP-FC cells provide a pathway by which drugs that target dopamine receptors for the treatment of neuropsychiatric disorders can act in the BG yet modulate activity in FC. The presence of a direct BG output to cortex extends the looped architecture through which the cortex-BG-thalamus control adaptive behavior and can become dysregulated to cause disease. Together our thesis results support the phenomenology of the BG pathway model, but suggest a major revision to the underlying circuitry.

<p> The research explores the functional architecture of the brain using fMRI in combination with behavioral and cognitive paradigms. Studying large-scale dynamic interactions of the brain's intrinsic connectivity networks (ICNs) forms the basis of the presented work. ICNs are networks of brain structures that display synchronous activity. Since the discovery of the default mode network in the resting brain, researchers have documented a handful of cortical, cerebellar and subcortical networks with a high level of similarity across subjects. There is striking convergence between ICNs observed in the resting brain and those elicited in activation tasks. ICNs can form when engaging in complex naturalistic tasks such as engaging in a simulated driving experience. ICNs can be identified using &ldquo;blind source separation methods&rdquo; such as Independent Components Analysis&mdash;a method well-suited for naturalistic and model-free activation-task designs. Most functional connectivity studies have examined the relationship between brain structures over extended periods of time. More recent work suggests that functional connectivity strength exhibits non-stationary fluctuations across longer time scales. Currently, little is known about how brain networks or ICNs interact during complex cognition or when externally engaged in a task. </p><p> Two experiments are presented along with the methods used to study large-scale network interactions while subjects are engaged in complex social cognition as well as basic oculo-motor function. Although the tasks differ in terms of the cognitive and perceptual processes involved, both experiments utilize a similar visually guided paradigm. This similarity enables us to study the effect of the task on the observed large-scale ICN functional interactions and to better our understanding of the functional significance of these functional dynamic organizations. Moreover, visually-guided paradigms can induce synchronous and unified experience across subjects, which allows us to study the common event-related and stimulus-driven interactions. Finally, large-scale network organization at various time scales is also discussed</p>

<p>Almost everyone will experience pain at some point in their lives. While pain is generally adaptive, and alerts the body to potential tissue damage, chronic pain is a disruptive disease with a huge psychological and economic cost. Of all the senses, the molecular basis of pain is perhaps the least understood. Without an understanding of the genes involved in pain, it is difficult to develop new drugs to treat pain. Current pain drugs are often ineffective at treating pain, especially chronic pain, and can cause many side effects. In this dissertation, I have further developed the Drosophila nociception paradigm. </p><p>First, I investigated the responses of Drosophila melanogaster larvae to a naturally occurring noxious stimulus, the parasitoid wasp. These wasps use a sharp ovipositor to lay their eggs inside of the larvae. The eggs hatch, and the wasp larva then eats the Drosophila larva from the inside, resulting in the death of the Drosophila. In response, Drosophila larvae have evolved multiple mechanosensory behaviors to fight attacks from wasps. The type of defensive behavior depends on the somatotopic location of the attack along the larval body wall, as well as the degree of penetration of the larval cuticle by the wasp ovipositor. I found that the class IV neurons, which are the larval nociceptors, and the pickpocket gene are important for mediating nocifensive responses to parasitoid wasp attacks.</p><p>While parasitoid wasps are the most ecologically relevant noxious stimulus for Drosophila larvae, the behavioral assays are time consuming and very low throughput. Thus, I utilized a thermal nociception assay in a genetic screen to discover new ion channels involved in the detection of noxious heat. In this assay larvae are touched lightly with a hot probe. In collaboration with Kia Walcott, I completed a forward genetic screen that knocked down 90% of the ion channels in the Drosophila genome. We found fourteen ion channel genes that are important for larval nociception. </p><p>The dendritic morphology of the nociceptor neurons is well studied, but the role of ion channels in governing the dendritic morphology had yet to be explored. We therefore screened the fourteen genes that we found to have a role in thermal nociception for a role in dendrite morphogenesis. Knockdown of six of the genes caused dendritic defects. These required genes represented a wide variety of transporters and channels, including potassium channels, TRP channels, and ligand gated channels. I also generated a genetic null mutant fly for coyotemint, an ABC transporter.</p><p>Lastly, I investigated the role of the calcium gated potassium channel, SK, in thermal nociception. Previous work in the lab had demonstrated that larvae null for SK exhibited a hypersensitive phenotype to a noxious thermal stimulus. I determined that one isoform of SK, SK-M, rescues this phenotype, and is necessary for thermal nociception. Additionally, I built an apparatus that allowed for the confocal imaging of genetically encoded calcium indicators while a larva undergoes a thermal ramp. This set-up allowed us to explore the role of SK in the physiology of the class IV neurons. We found the larvae null for SK exhibit increased levels of calcium during a thermal ramp.</p><p>In conclusion, my work has explored both the ethology of nociception in Drosophila larvae, as well as engaged in the search for new genes involved in nociception.</p> / Dissertation

Thesis (Ph. D.)--University of Toronto, 2008. / Includes bibliographical references.

Prenatal protein malnutrition continues to be a significant problem in the world today. Exposure to prenatal protein malnutrition increases the risk of a number of neuropsychiatric disorders that are associated with inhibitory interneurons, including depression, schizophrenia and attention deficit hyperactivity disorder. Previous studies have found that neurons in anterior cingulate and medial prefrontal regions respond excessively to restraint stress in prenatally malnourished rats. In this study, we investigate if prenatal protein malnutrition affects inhibitory the subpopulation of interneurons in the prefrontal cortex in relationship to the higher initial stress response. This was done using double-labeling immunohistochemistry with c-Fos to mark activated neurons and parvalbumin to mark inhibitory interneurons. Numbers of single and double-labeled neurons were quantified with unbiased stereology. Statistical analysis demonstrated that there was no effect of prenatal malnutrition on the total number of neurons or on the number of parvalbumin neurons. However, prenatal malnutrition was associated with a significant increase in the number of inhibitory parvalbumin positive neurons activated by restraint stress. This suggests that prenatal malnutrition altered the excitability of these inhibitory interneurons either directly or by altering their connectivity.

The process of learning contains multiple aspects, whose intricacies have yet to be fully understood. The current experiment utilized gaze tracking technology to observe whether variables in subjects' gaze (e.g total time spent on a given image, percentage of time spent on cognitively salient features of the image) was predictive of the subject learning the material. Subjects consisted of students from a Medical Gross Anatomy course, who were tested twice-once before they had learned the course and once after they had learned a certain amount of material. Following the baseline testing, subjects were broken up into three groups, A, B and C, each representing a later visit in the course. Results indicated that groups B and C tended to spend more time on cognitively salient areas of interest, but this was moderated by familiarity. Moreover, results indicated that groups B and C tended to spend more time on images overall (end time) compared with group A or the baseline group. Overall, the results obtained were ambiguous, and warrant further study in order to arrive at a clear conclusion. Future directions of study may want to consider other gaze tracking variables, such as the time at which a subject first looks at a cognitively salient area of interest.

The amygdala and orbitofrontal cortex are critical sites for processing emotional content. The amygdala sends dense pathways preferentially to the posterior orbitofrontal cortex (pOFC) and to the magnocellular part of the mediodorsal thalamic nucleus (MDmc), which is itself robustly connected with pOFC. This tri-partite circuit is thought to be activated when associating stimuli with emotional value, and is necessary to flexibly adapt behavior to changing circumstances, but its features and synaptic interactions are unknown. Labeling of pathways with distinct neural tracers in rhesus monkeys revealed that amygdalar terminals in pOFC were denser and larger compared to those in other prefrontal cortices. Further, amygdalar terminals in pOFC were even larger than thalamocortical terminals, which are considered highly efficient drivers of cortical neurons. In comparison with thalamocortical pathways, amygdalar terminals innervated more excitatory neurons and were more frequently multisynaptic. These features suggest that the amygdala sends a highly efficient excitatory pathway to pOFC. Among a small proportion of innervated inhibitory neurons, the pathway from the amygdala to pOFC preferentially targeted the neurochemical classes of calbindin and calretinin inhibitory neurons in the upper layers, which are functionally suited to suppress distracting stimuli and enhance relevant signals. Further, the amygdalar pathway to MDmc targeted thalamocortical relay neurons, including those that project to pOFC, providing a second route for amygdalar signals to reach cortex. Neurochemical and morphological differences among terminals suggest that the direct pathway from the amygdala to pOFC and the indirect route through MDmc arise from separate neuronal populations in the amygdala. In MDmc, axon terminals from the amygdala formed synaptic triads, a thalamic specialization connecting excitatory projection neurons and local inhibitory neurons. This synaptic specialization is akin to what is found in sensory thalamic nuclei connecting peripheral sensory afferents with cortex. By analogy, the amygdala may act as a sensor of affective value, relaying signals about internal states to cortex through MDmc. The synaptic specializations shown here in the circuit that tightly interlinks the amygdala, MDmc, and pOFC shed light on the functional circuitry for emotional behavior and its disruption in psychiatric disorders such as phobias and obsessive compulsive disorder.

Rcc2 is a Rac guanine nucleotide exchange factor recently identified as a principal signaling component of integrin adhesion complexes that also plays a central role in the completion of mitosis and cytokinesis. Rcc2 mRNA is enriched in a class of neural progenitors in the ventricular zone, short neural precursors. Although Rcc2 mRNA is present at high levels in the ventricular zone during neurogenesis, the impact of Rcc2 on cortical development has not been previously studied. We used two methods to study the role of Rcc2 in vivo. First we isolated a portion of the upstream regulatory region of Rcc2 and used it to express a fluorescent protein. Additionally, we used an shRNA targeting Rcc2 to knockdown expression of Rcc2. We found that the promoter region of Rcc2 labeled cells that were near the board of the ventricular zone and subventricular zone, and tended to be positive for Sox2 but not Tbr2, when compared to the general progenitor population. Progenitors electroporated with Rcc2 shRNA were closer to the ventricular surface than those with functioning Rcc2. Examination of the cell cycle in cells electroporated with Rcc2 shRNA indicated no difference to those with Rcc2. We found that Rcc2 was active during neurogenesis in ventricular zone progenitors. Additionally, our analysis shows that Rcc2 may be involved in the migration of progenitors during neurogenesis. Further works needs to be done to further elucidate the role of Rcc2.