The spatial arrangement of neuronal responses in primary auditory cortex (A1) has so far been investigated by using microelectrode recording techniques or imaging of the intrinsic signal, which led to controversial results, at present still discussed. On the other hand, two-photon calcium imaging allows us to investigate the cortical functions at an unprecedented level of spatial detail, and has recently offered new insight into the fine-scale organization of frequency responses in A1. In this thesis, I used two-photon calcium imaging to compare, for the first time, the fine-scale cortical representation of sound frequency to that of two other sound features, crucial for survival and communication in all mammals: differences in intensity between the two ears (interaural level differences; ILDs), and frequency modulation (FM). I found that most neurons in layers II-III of the mouse A1 were tuned to ILDs favouring the contralateral ear, but midline and ipsilateral tuning were present too. Binaural preferences were heterogeneously distributed in space, both on the fine scale (within ∼ 200 μm) and on the global one (up to ∼ 1 mm). Moreover, A1 neurons were mostly tuned to slow FM sweeps within the range of those used in species-specific calls. Cells activated by similar rates tended to be spatially proximal, indicating a level of local organization similar to the one I found for frequency tuning, and higher than that of ILD responses. Finally, I set the groundwork for two-photon studies of the A1 of the ferret, by presenting the first evidence of the microscopic organization of the tonotopic map in this species. My results shed light on some long-held questions about the response properties of A1, and confirm two-photon imaging as a powerful tool for investigating the processing of sensory signals in the cortex of both small and large mammals.

<p>Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.</p> <p>In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18426号 / 生博第306号 / 新制||生||40(附属図書館) / 31284 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松崎 文雄, 教授 渡邉 大, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The locus coeruleus (LC) projects ubiquitously to the cortex, and noradrenaline (NA) exerts powerful neuromodulatory control on cortical excitation and inhibition. Previous work has shown that NA plays an important role in motor processes, and further posits that dysregulation in NA function could be one of the culprits of motor-related deficits in many neurodevelopmental disorders, including Autism Spectrum Disorder. In order to characterize the change in NA levels during motor learning in awake and behaving mice, I employed a newly developed optical NA sensor, combined with in vivo two-photon imaging, to visualize spatiotemporal activation patterns of NA in the motor cortex. This experimental approach allows us to track and chronically image the same region of the motor cortex over multiple days, thus permitting the characterization of NA activity throughout the entirety of the motor learning process. I found that NA levels increase significantly during the initial phase of learning, which coincides with the structural and functional plastic changes that have been previously reported in the motor cortex during early stages of motor learning. The NA activity returns to baseline levels as the mice develop their movement strategy; however, the regions of NA release become more spatially clustered during the learning process. The results reported in this thesis provide a novel glimpse into the dynamics of NA activity in the motor cortex during motor learning, and it will provide new direction for the development of therapeutic strategies and diagnostic criteria for motor-related dysfunction in neurodevelopmental diseases.