Many basal forebrain and mesopontine tegmental cholinergic projection systems tend to overlap in their origins. This raises the possibility that these projection systems are collateralized to innervate divergent areas. In experiment one, the degree to which basal forebrain and mesopontine tegmental neurons that innervate the reticular thalamic nucleus have axons that collateralize to innervate the cortex as well was examined with a retrograde fluorescence labeling method combined with immunohistochemistry. A significant portion of the labeled neurons in the region of the nucleus basalis magnocellularis and pedunculopontine tegmental nucleus projecting to the reticular thalamic nucleus were observed to be also labeled (double-labeled) following intracortical tracer injections. Many of these double-labeled neurons displayed choline acetyltransferase choline acetyltransferase immunoreactivity. It was also shown that numerous basal forebrain neurons that innervated the reticular thalamic nucleus contained the calcium-binding protein, parvalbumin. These neurons tended to be located more rostrally than the ChAT immunoreactive neurons; primarily in the region of the ventral pallidum. There was some indication that parvalbumin-containing neurons in the basal forebrain that innervate the reticular thalamic nucleus also have axons that branch to innervate the cortex. Finally, none of the basal forebrain neurons innervating the reticular thalamic nucleus was found to contain somatostatin. In experiment two, the degree to which basal forebrain neurons have axons that collateralize to innervate the interpeduncular nucleus and hippocampus was examined with retrograde fluorescence labeling methods. Labeled neurons projecting to both of these limbic structures were observed only occasionally. Comparison of the distribution of single labeled neurons innervating each of these structures revealed that within the region of origin, in the horizontal limb of the diagonal band, neurons innervating the interpeduncular nucleus tended to be located dorsally to those innervating the hippocampus. The results of these experiments are discussed in relation to their anatomical and functional implications toward a greater understanding of the basal forebrain and mesopontine cholinergic and non-cholinergic projection systems. / Medicine, Faculty of / Graduate

Le facteur XII (FXII) est une sérine protéase de 80 kDa produite et sécrétée par le foie qui initie la voie intrinsèque de la coagulation. Diverses études ont montré un rôle délétère du FXII dans des pathologies cérébrales telles que l'accident vasulaie cérébral, la maladie d'Alzheimer et la slérose en plaques. Néanmoins, l'impact direct du FXII sur le devenir des cellules neuronales reste inconnu. De plus, l'expression du FXII dans le système nerveux central (SNC) n'a pas encore été étudiée. Le premier objectif de nos travaux a été d'étudier le rôle du FXII dans l'apoptose neuronale puis dans un second temps d'étudier l'expession du FXII dans le SNC. Dans notre étude, nous avons constaté que le FXII protège les neurones en culture de la mort apoptotique. Les effets bénéfiques du FXII résultent de l'interaction directe du FXII avec le récepteur du facteur de croissance épidermique (EGFR). L'activation de l'EGFR par le FXII déclenche les voies signalétiques antiapoptotiques MAPK. Il est intéressant de noter que la forme double chaîne du FXII, αFXIIa, exerce des effets protecteurs complémentaires en convertissant le précurseur du facteur de croissance hépatocytaire en sa forme mature, ce qui active à son tour le récepteur MET. Enfin, dans notre étude, nous avons observé que le FXII était exprimé dans le SNC (à la fois l'ARNm et la protéine). Cette expression est notamment retrouvée au niveau des neurones corticaux. Ce travail décrit un nouveau mécanisme d'action du FXII et décrit les neurones en tant que cellules cibles pour les effets facteur de croissance du FXII. Dans l'ensemble, ces travaux devraient donc aider à mieux comprendre comment le FXII agit dans les pathologies cérébrales en tant que sérine-protéase unique à l'interface de la thrombose, de l'inflammation et de la survie cellulaire. / Factor XII (FXII) is an 80 kDa serine protease produced and secreted by the liver that participates in the intrinsic coagulation pathway. Various studies have shown a deleterious role of FXII in cerebral pathologies like stroke, Alzheimer disease and multiple sclerosis. Nevertheless, the direct impact of FXII on neuronal cell fate remains unknown. In addition, the expression of FXII in the central nervous system (CNS) has not been investigated yet. The first objective of our work was to study the role of FXII in neuronal apoptosis and then to study the expression of FXII in the CNS. In our work, we found that FXII protects cultured neurons from apoptotic death by a growth factorlike effect. The beneficial effects of FXII result from the direct interaction with the epidermal growth factor receptor (EGFR). Activation of EGFR by FXII triggers antiapoptotic signaling MAPK pathways. Iteestigl, the to hai fo of FXII, αFXIIa, eets opleeta potetie effets oetig the hepatocyte growth factor (HGF) precursor into its mature form, which in turn activates MET receptor. Finally in our work we observed FXII expression in the CNS (both mRNA and protein). This expression is particularly found in cortical neurons. This work describes a novel mechanism of action of FXII and discloses neurons as target cells for growth factor effects of FXII. Overall, these works should thus help further understanding how FXII acts in brain diseases as a unique serine-protease at the interface of thrombosis, inflammation and cell survival.

Animals must respond to external cues and changes in internal state by modifying their behavior. The basal ganglia are a collection of subcortical nuclei that contribute to action selection by integrating sensorimotor, limbic and reward information to control motor output. In early life, however, animals display distinct behavioral responses to risk and reward and enhanced vulnerability to neuropsychiatric disease. This arises from the postnatal maturation of brain structures such as the striatum, the main input nucleus of the basal ganglia. Here, using biochemical, electrophysiological and behavioral approaches in transgenic mice, I have explored the molecular and circuit mechanisms that control striatal maturation. In Chapter 1, I begin by reviewing the structure, physiology and function of the basal ganglia, with an emphasis on the striatum. I then describe the existing literature on the development and maturation of striatal neurons and their afferents. In Chapter 2, I review the molecular mechanisms of macroautophagy, a lysosomal degradation pathway that has recently been implicated in the regulation of neurotransmission, including its contribution to neuronal development, neurotransmitter release, and postsynaptic function. The subsequent chapters can be split into two themes. In the first, encompassing chapters 3 and 4, I characterize the postnatal maturation of striatal physiology and define circuit mechanisms that control this process. In Chapter 3, I demonstrate that dopamine (DA) neurotransmission in the striatum initiates the maturation of striatal projection neuron (SPN) intrinsic excitability. I show that DA signaling leads to the maturation of SPN excitability via increased activity of the potassium channel, Kir2. Interestingly, introduction of DA beginning in adulthood could not rescue SPN hyperexcitability while it could during the juvenile period. In Chapter 4, I characterize the maturation of cholinergic interneurons (ChIs) in the striatum and describe the biophysical mechanisms that drive increases in spontaneous activity that occur in ChIs during postnatal development. Finally, I show that the functional maturation of ChIs leads to changes in DA release during the postnatal period. The second theme includes Chapters 5 and 6, in which I explore the role of macroautophagy in striatal function and development. In chapter 5, I used biochemical approaches to show that autophagic flux is suppressed postnatally in the striatum due to increased signaling through the kinase activity of the mammalian target of rapamycin. In Chapter 6, I generated conditional knockouts of Atg7, a required macroautophagy gene, in different populations of SPNs and find that macroautophagy plays cell-type specific roles in SPN physiology. In one subtype of SPNs, macroautophagy regulates intrinsic excitability via degradation of Kir2 channels, which is the first demonstration of macroautophagic control of neuronal excitability. Finally, in Chapter 7, I conclude with a general discussion, where I highlight themes in the molecular and circuit mechanisms of striatal maturation and their implication for neurodevelopmental disease.

The encoding of olfactory information starts from the interaction between odorant molecules and olfactory sensory neurons (OSNs). In mouse, one mature olfactory sensory neuron (OSN) almost exclusively expresses one out of ~1,000 odorant receptors (ORs). The relationship between odorants and ORs is promiscuous: one odorant can activate multiple ORs and one OR can be activated by many odorants. This combinatorial olfactory coding scheme is fundamental, but not sufficient to fully understand the peripheral encoding of odor mixtures. Almost all naturally-occurring smells consist of many different odorous compounds; for example, the perception of rose is composed of (-)-cis-rose oxide, beta-damascenone, bata-ionone and many other odorants. It is well appreciated in psychology and perfumery that different components in an odor blend can affect each other, producing modulation effects. However, these effects are often considered to be the results of higher center processing, while odor interactions at the peripheral level have not been comprehensively measured. To evaluate peripheral neuronal responses to odor blends, it is necessary to profile the response patterns of a large population of OSNs while the responses of each individual OSN can be resolved. Conventionally, this has been achieved by imaging OSNs acutely dissociated from the olfactory epithelium with a regular epi-fluorescent microscope. In Chapter 2 of this thesis, such method was utilized to characterize the response patterns of three groups of bio-isosteres. This study reveals that OSNs discriminate odors primarily based on their topological properties rather than chemical properties. Chapter 3 investigates the modulation effects of Hedione, a chemical that has been widely used in perfumery for 60 years. Hedione is psychophysically known as an enhancer that brings up the volume of floral and citrus odors, but the underlying mechanism remains largely unknown. Our study showed that Hedione could both enhance and inhibit odor responses in peripheral neurons, with inhibition being the dominant effect. Moreover, dose-dependent analyses have shown that odorant receptors with lower binding affinity are more prone to inhibition, leading to the hypothesis that Hedione may act as a weak antagonist, which highlights the scent of the leading compound through contrast enhancement. However, the cell imaging method in Chapter 2 and 3 was limited by the low throughput (200 cells per field of view) and cell damage during digestion. Utilizing a new advance in microscopy, Swept Confocally Aligned Planar Excitation (SCAPE), I was able to perform 3D volumetric imaging on the intact olfactory epithelium of OMP-CRE+/-GCaMP6f-/- mice with a perfused half-head preparation. This method is capable of recording over 10,000 OSNs simultaneously with high spatial and temporal resolution. The process of establishing the imaging protocol and data analysis pipeline has been detailed in Chapter 4. Chapter 5 characterizes OSN responses to odor blends using the SCAPE microscopy. A large number of responding cells showed inhibited or enhanced responses to odor mixtures compared with responses to each individual component. Eight structurally and perceptually distinct chemicals were tested, all shown to act as antagonists or enhancers to some extent. Compared with a monotonically additive coding scheme, the presence of widespread modulation effects could diversify the output, thereby increasing the capacity of the olfactory system to distinguish complex odor mixtures. Taken together, these results show that olfactory information is subject to widespread modulation in the olfactory epithelium. This unusual complexity at the primary receptor level implies an information coding strategy different from those utilized by visual and acoustic systems, where complex interactions among stimuli only occur at higher levels of processing. Further experiments are needed to explain the mechanisms at the molecular level and to link peripheral neuronal responses to psychophysics and behavior.

Behaving in a complex world requires flexible mapping between sensory inputs and motor outputs. One must be able to make decisions about what actions to take based on a wide variety of inputs. This presents a routing problem: brain areas involved in decision making must receive information encoded by different sensory neurons in different situations. In this thesis I investigate this routing problem using two variations of the random dot motion task which require flexible routing. In the first, a single random dot motion task appears in different locations on different trials. Recording from the lateral intraparietal area (LIP) revealed several neural features which varied with stimulus location. A second task made it possible to disentangle routing from other signals, by separating the time of routing from the onset of motion and decision making. In this second task, a visual cue indicated the location at which relevant motion would appear. After the cue was extinguished, two random dot motion patches appeared. An informative patch appeared at the cued location, and an uninformative patch appeared at another location. Comparison of these two tasks revealed three location dependent signals at motion onset: a visual signal related to surround suppression, a second suppressive signal that may set the amount of evidence required for decision making, and a 12-20 hertz oscillation in firing rate. This oscillation appears to be a signature of flexible information routing. It appears at motion onset when the motion stimulus varies in location unpredictably; it appears at cue onset when a spatial cue indicates the location information must be routed from; and it does not appear when stimulus location is fixed and flexible routing is not required. Future work on this project will eventually require tools which are not well developed for use in rhesus macaques. The final chapter describes two projects which attempt to address this problem, one through the use of optogenetics in monkeys and the other by adapting an established monkey behavioral task for use in mice.

Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, is widely expressed in various systems, including the immune and nervous systems and plays a critical role in controlling innate and adaptive immune responses. We previously reported that opioids inhibit cell growth and trigger apoptosis. However, the underlying mechanism by which TLR2 mediates apoptosis in response to opioids is not yet known. Here we show that chronic morphine treatment in primary neurons dramatically increased the expression of TLR2 at both the messenger RNA and protein levels. In addition, TLR2 deficiency significantly inhibited chronic morphine-induced apoptosis in primary neurons. Activation of caspase-3 after morphine treatment is impaired in TLR2 deficient primary neurons. Moreover, morphine treatment failed to induce an increased level of phosphorylated glycogen synthase kinase 3 beta (GSK3β) in TLR2 deficient primary neurons, suggesting an involvement of GSK3β in morphine-mediated TLR2 signaling. These results thus demonstrate that opioids prime neurons to undergo apoptosis by inducing TLR2 expression. Our data suggest that inhibition of TLR2 is capable of preventing opioids-induced damage to neurons.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the rat central nervous system, notably in areas involved in control of autonomic and neuroendocrine functions. The aim of this study was to evaluate the effects of CART peptide fragment 55-102, referred to herein as CARTp, by intrathecal injection on blood pressure (BP) and heart rate (HR) before and after intrathecal glutamate in urethane-anesthetized male Sprague-Dawley rats. CARTp (0.1-10 nmol) administered intrathecally caused no or a small, statistically insignificant increase of blood pressure and heart rate, except at the concentration of 10 nmol, which caused a significant increase of blood pressure and heart rate. Intrathecal glutamate (0.1-10 nmol) produced a dose-dependent increase in arterial pressure and heart rate. Pretreatment with CARTp dose-dependently potentiated the pressor effects of glutamate (1 nmol), which by itself elicited a moderate increase of blood pressure and heart rate. Further, CARTp significantly potentiated the tachycardic effect of glutamate at 1 and 5 nmol, but attenuated the response at 10 nmol. The effect of CARTp was long-lasting, as it enhanced glutamatergic responses up to 90 min after administration. Prior injection of CARTp antiserum (1:500) but not normal rabbit serum nullified the potentiating effect of CARTp on glutamatergic responses. The result suggests that CARTp, whose immunoreactivity is detectable in sympathetic preganglionic neurons as well as in fibers projecting into the intermediolateral cell column, augments spinal sympathetic outflow elicited by glutamate at lower concentrations and may directly excite neurons in the intermediolateral cell column at higher concentrations.

The taste of sugar is one of the most basic sensory percepts for humans and other animals. Remarkably, animals can develop a strong preference for sugar even if lacking a functional sweet taste receptor, pointing to a detection mechanism independent of the sense of taste. Here we examined the neural basis for sugar preference and demonstrate that a population of neurons in the brainstem are activated via the gut-brain axis to create preference for sugar. These neurons are stimulated in response to sugar, but not to artificial sweeteners, and are activated by direct delivery of sugar into the gut. We demonstrate that these cells receive direct vagal inputs, which are necessary for their response to sugar. Using functional imaging we monitored the activity of the gut-brain axis, and identified the vagal neurons activated by intestinal delivery of glucose. We characterized the nature of their responses, establish their specificity, and identify the mechanism required for sugar sensation. Finally, we engineered animals where synaptic activity in this gut-to-brain circuit was genetically silenced, and prevented the development of a behavioral preference for sugar. Together, these findings reveal a gut-to-brain post-ingestive sugar-sensing pathway critical for the development of sugar preference. In addition, they explain the neural basis for the behavioral differences of sweeteners versus sugar, and uncover an essential circuit underlying sugar’s highly appetitive effects.

As the prevalence of Parkinson’s disease is expected to increase gradually over the years based on recent scientific predictions, developing a treatment plan to mitigate the development of this disease is essential. Previous research tried to tackle the motor and non-motor symptoms associated with the disease. That said, some symptoms seem to persist, and the quality of life of PD patients continues to decline. Zebrafish have emerged as a strong model to study the regeneration of DAergic neurons as they have the ability to show robust adult neurogenesis. Here, we used adult zebrafish to investigate DAergic neuron regeneration following ablation in various brain regions. In addition, we tested the efficacy of Nifurpirinol, an alternative substrate to MTZ, in ablating DAergic neurons in the adult zebrafish brain. Lastly, we tracked how the ablation of DAergic neurons influences the motor activity of adult zebrafish and how they tend to recover over time. Results showed a significant reduction in DAergic neurons at 7 days following the MTZ treatment in the olfactory bulb, telencephalon, and the periventricular pretectal nucleus. NFP also caused similar changes, albeit they were less statistically significant. In response to ablated DAergic neurons, MTZ-treated fish showed a significant increase in the number of neural stem cells undergoing proliferation at 1 dpt. However, the highest spike in proliferative cells, especially neural stem cells, was found at 7 dpt. This time point corresponded with the greatest decrease in DAergic neurons following ablation. These cellular changes were observed in the olfactory bulb and the telencephalon. That said, more drastic changes were noticed in the rostral and medial telencephalon. Results also showed that the adult zebrafish brain was not able to significantly replenish the number of DAergic neurons as early as 15 dpt. Based on previous observations, it seems that adult zebrafish need at least 45 days to regenerate their DAergic neurons to levels comparable to the DMSO control. Lastly, behaviour analysis showed that NFP has the most significant impact on motor activity across three different parameters at 0 hpt. MTZ also had similar effects on motor activity; however, it was less pronounced. The impact on the behaviour level seems more transient as some recovery was observed at 7 dpt. Overall, this transgenic zebrafish line allowed us to explore how and when the adult zebrafish brain was able to efficiently recover following the specific ablation of DAergic neurons. In addition, it expanded our understanding of adult neurogenesis which will hopefully allow us to better approach patients with Parkinson’s disease.

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