Adult neurogenesis of olfactory receptor neurons (ORNs) occurs in diverse organisms including in decapod crustaceans. This dissertation describes the molecular, cellular, and physiological changes that occur during adult neurogenesis of ORNs in the antennular lateral flagellum (LF) of the spiny lobster Panulirus argus. Examination of the role of splash (spiny lobster achaete scute homolog) in adult neurogenesis and regeneration using in situ hybridization showed splash was not closely associated with the formation of sensory neurons under normal physiological conditions. Damage to the LF, which induces regeneration, enhanced splash expression, suggesting an association between splash with regeneration and repair. This study suggests that splash plays multiple roles in the olfactory organ of adult spiny lobsters. Examination of extracellular and intracellular Ca2+ in mediating spontaneous and odor-induced responses of ORNs, using calcium imaging showed that odor-induced Ca2+ transient responses and spontaneous Ca2+ oscillations in ORN somata are primarily mediated by an influx of extracellular Ca2+ through Co2+ -sensitive Ca2+ channels, but that intracellular Ca2+stores also have some contribution. These responses are independent of TTX-sensitive Na+ channels, suggesting that these Ca2+ responses may reflect receptor potentials. Examination of changes in odor specificity, sensitivity, and temporal responses in adult-born ORNs showed an increase in the percentage of odorant-responsive ORNs as they age from newly-born cells to mature, and a decrease in odorant-responsive ORNs as they senesce. As adult-born ORNs age, there was a decrease in the percentage of ORNs that undergo spontaneous Ca2+ oscillations and an increase in the amplitude of oscillation. ORNs became more broadly tuned as they senesce, and their response profile, defined by the most effective odorant, changed. Odor sensitivity changed with age. This study demonstrated that the physiological response properties of adult-born ORNs changed with functional maturation. Taken together, this dissertation reveals molecular, cellular and physiological changes in adult born ORNs and elucidates mechanisms of adult neurogenesis.

Sensory signals are represented and propagated as spiking activity in multiple neuronal populations to lead to cognitive or motor behavior in organisms. Neural processing underlying sensory-motor behavior is understood by uncovering the governing computational principles and the biophysical mechanisms that implement the principles. While these mechanisms have been studied extensively at the single-neuron and system levels, activity within neuronal networks significantly impact neural processing. For example, there are spatiotemporal interactions (neural correlations) between responses of neurons within populations that could potentially impact signal representation and propagation. Furthermore, the effects of associative plasticity are also expected to alter network activity and its propagation. The effects of plasticity on network activity cannot be predicted from individual neuronal responses due to the complex, non-linear interactions within neuronal networks. Thus examining neural correlations in network activity and the propagation of network activity, requires recording spiking activity from large, heterogeneous, populations of spatially distributed neurons simultaneously. Studies addressing the propagation of network activity have been limited to theoretical approaches. Empirical studies have been limited by the technical difficulties in recording from a large number of neurons simultaneously. To overcome this challenge we developed a novel technique, dithered random-access functional calcium imaging. This imaging technique records and extracts suprathreshold activity from a large number of neurons. This technique also has a high spike detection efficiency and millisecond temporal precision. We applied this technique to measure the propagation of activity and neural correlations in activity evoked by afferent, thalamocortical inputs in the recurrent cortical networks of the mouse barrel cortex. We found that the cortical activity evoked by novel (naïve), thalamocortical inputs showed limited propagation of activity and decrease in propagation of neural correlations (measured from neuronal pairs within each population) from L4 to L2/3 network of the responding column. However, associative cortical plasticity was induced from pairing thalamocortical inputs with intracortical inputs. This pairing resulted in increased propagation of activity. The pairing also modified the propagation of neural correlations. Our results suggest that synaptic plasticity in intracortical circuits contributes to the modified propagation of activity and neural correlations. The modified propagation of neural correlations could in turn contribute to behavioral performance in vivo following perceptual learning. / text

Das Geruchssystem ist in der Lage, mittels sogenannter kombinatorischer Kodierung einen hochdimensionalen Geruchsraum durch eine begrenzte Anzahl von olfaktorischen Rezeptorneuronen (ORN) abzutasten. Hierbei weisen verschiedene ORN-Klassen eine breite und gleichzeitig spezifische Geruchssensitivität auf, durch welche ein geruchsspezifisches Antwortmuster auf Populationen von Mitral-/Tufted Zellen (M/T) des bulbus olfactoris (OB) abgebildet wird. Neueren Untersuchungen zufolge sind diese Antwortmuster nicht notwendigerweise statisch, sondern enthalten Information in ihrer zeitlichen Entwicklung. Im OB von Larven des Krallenfrosches Xenopus laevis wurde herausgefunden, dass sowohl Geruchsidentität als auch -Konzentration besser vorhergesagt wird durch M/T Antwortlatenzmuster als durch durchschnittliche Feuerraten. Diese Arbeit befasst sich mit der Messung von ORN-Aktivität auf verschiedenen raumzeitlichen Skalen. Auf der Ebene von ORN Populationen wurde mit Hilfe von konfokaler Mikroskopie und [Ca2+] -sensitiven Fluoreszenzfarbstoffen untersucht, in wie weit Latenzmuster auftreten. Es wurde gezeigt, dass Latenzmuster im Unterschied zu M/T Zellen eine geringere Vorhersagekraft für die Geruchsstoffkonzentration besitzen als Feuerratenmuster. Außerdem wiesen Ensemble-Feuerraten einen größeren dynamischen Bereich bezüglich der Geruchsstoffkonzentration auf als Latenzen. Durch eine Kombination von schneller (1,25 kHz) [Ca2+] -Bildgebung und whole-cell Patch-Clamp Technik in einzelnen ORNs wurde die zeitliche Entwicklung der dreidimensionalen intrazellulären Ca2+ -Konzentration während eines Depolarisationspulses gemessen. Mit Hilfe von pixelweiser Angleichung eines numerischen Modells wurden Ballungen spannungsabhängiger Ca2+ Kanäle (VGCC) auf der Oberfläche von ORN-Somata lokalisiert. Da der durchschnittliche gemessene VGCC-Kalziumioneneinstrom einen geringen Beitrag im Vergleich zum Ca2+ Generatorstrom darstellt (<80 pA bzw. geschätzt 900 pA), erklärte sich, warum einzelne Aktionspotentiale nicht mittels [Ca2+] Bildgebung gemessen werden konnten. Bezüglich VGCC-Häufung und möglicher Kolokalisation mit Kaliumkanälen hoher Leitfähigkeit (BK) wurde der Effekt von BK Blocker Iberiotoxin auf ORN-Reizantworten untersucht. In einer Untergruppe aller ORNs wurde eine Verringerung der Antwortamplituden nach Anwendung von Iberiotoxin festgestellt. Aus den gezeigten Ergebnissen wurde geschlossen, dass eine wichtige Funktion von Glomeruli im OB die Konversion von Geruchsinformation zwischen Feuerratenkodierung und Latenzkodierung sein müsse.

No description available.

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.

The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada

No description available.

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

The understanding and treatment of brain disorders as well as the development of intelligent machines is hampered by the lack of knowledge of how the brain fundamentally functions. Over the past century, we have learned much about how individual neurons and neural networks behave, however new tools are critically needed to interrogate how neural networks give rise to complex brain processes and disease conditions. Recent innovations in molecular techniques, such as optogenetics, have enabled neuroscientists unprecedented precision to excite, inhibit and record defined neurons. The impressive sensitivity of currently available optogenetic sensors and actuators has now enabled the possibility of analyzing a large number of individual neurons in the brains of behaving animals. To promote the use of these optogenetic tools, this thesis integrates cutting edge optogenetic molecular sensors which is ultrasensitive for imaging neuronal activity with custom wide field optical microscope to analyze a large number of individual neurons in living brains. Wide-field microscopy provides a large field of view and better spatial resolution approaching the Abbe diffraction limit of fluorescent microscope. To demonstrate the advantages of this optical platform, we imaged a deep brain structure, the Hippocampus, and tracked hundreds of neurons over time while mouse was performing a memory task to investigate how those individual neurons related to behavior. In addition, we tested our optical platform in investigating transient neural network changes upon mechanical perturbation related to blast injuries. In this experiment, all blasted mice show a consistent change in neural network. A small portion of neurons showed a sustained calcium increase for an extended period of time, whereas the majority lost their activities. Finally, using optogenetic silencer to control selective motor cortex neurons, we examined their contributions to the network pathology of basal ganglia related to Parkinson’s disease. We found that inhibition of motor cortex does not alter exaggerated beta oscillations in the striatum that are associated with parkinsonianism. Together, these results demonstrate the potential of developing integrated optogenetic system to advance our understanding of the principles underlying neural network computation, which would have broad applications from advancing artificial intelligence to disease diagnosis and treatment.

No description available.