The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.

The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19765号 / 農博第2161号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4981(農学部図書室) / 32801 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 田川 正朋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The 129S2 inbred mouse strain is often used as a background strain in the production of genetically altered mice (i.e. knockout and transgenic mice). It is important to establish the behavioral phenotype of wild-type mice before making comparisons to genetically altered mice. Also, those comparisons can assist in the evaluation and interpretation of the in vivo effects of drugs. The drug discrimination assay measures the subjective effects of drugs and provides a measure of underlying neuropharmacological mechanisms responsible for the discriminative stimulus properties of drugs. The present study established the atypical antipsychotic drug clozapine as a discriminative stimulus in male 129S2 inbred mice and compared clozapine’s discriminative stimulus properties in 129S2 mice to C57BL/6 and DBA/2 inbred mice. By comparing the discriminative stimulus properties between inbred strains of mice we hope to obtain a fuller picture of the underlying neuropharmacological mechanisms of antipsychotic drugs.

Schizophrenia is a debilitating mental disorder that causes a large economic burden and is prevalent across all cultures and countries around the world. Although both environmental factors and genetics are known to play an important role in the etiology of schizophrenia, the exact role of genetics and its interaction with environmental factors in an individual’s predisposition to develop schizophrenia is poorly understood. Schizophrenia is characterized by symptoms that include positive symptoms (e.g. delusions, hallucinations, disorganized thinking and speech), negative symptoms (e.g. avolition, anhedonia, depressive-like behavior), and cognitive dysfunctions (e.g. executive functioning deficits in learning and memory, attention, and vigilance). Genomic screening has identified polymorphisms of the vesicular monoamine transporter 1 (VMAT1) gene (SLC18A1) that are associated with schizophrenia and bipolar disorder. The current study represents the first extensive phenotyping of both young and aged mice in which the VMAT1 gene (SLC18A1) has been deleted. The results demonstrated behavioral effects of deleting the VMAT1 gene that may relate to aspects of schizophrenic-like behavioral changes in this model. Specifically, young VMAT1 knockout mice displayed significant deficits in sensorimotor gating in the prepulse inhibition (PPI) task and in the acquisition of operant learning in the autoshaping task. When exposed to a mild stressor (24 hours of food deprivation), young VMAT1 knockout mice displayed a significant reduction in locomotor activity that was not evident under free-feeding conditions. Thus, young VMAT1 knockout mice showed deficits in tasks that model positive symptoms and cognitive deficits seen in schizophrenia; however, they did not display differences in behaviors related to models of the negative symptoms of schizophrenia or deficits in tasks designed to measure motor skills. While less extensive phenotyping was conducted in aged VMAT1 knockout mice, there were no significant deficits evident in any of the assays conducted in older animals. These findings demonstrated that deletion of the VMAT1 gene has behavioral effects that appear to be mediated by changes in brain monoamine function and changes in response to stressors (i.e. food deprivation) that may reflect changes in adrenal gland monoamine function.

Der ischämische Schlaganfall ist global eine der bedeutendsten Volkskrankheiten. Die derzeit verfügbaren kurativen Therapieoptionen werden vorrangig durch ein enges therapeutisches Zeitfenster limitiert. Ziel der aktuellen Schlaganfallforschung ist die Entwicklung von über dieses Zeitfenster hinaus wirksamen Therapien. Ein vielversprechender neuer Ansatz ist die experimentelle Behandlung mit humanen Nabelschnurblutzellen. Diese Arbeit erforscht das therapeutische Zeitfenster für die systemische Therapie des ischämischen Schlaganfalls mittels mononukleärer Nabelschnurblutzellen (hUCB MNC) in spontanhypertensiven Ratten nach permanentem Verschluss der Arteria cerebri media (pMCAO). Hierzu wurden die Therapiezeitpunkte 4, 24, 72, 120 Stunden und 14 Tage nach experimentellem Schlaganfall in einem komplexen Studiendesign inklusive neurofunktioneller Tests, magnetresonanztomographischer und immunhistochemischer Verfahren untersucht. In vitro wurde der Einfluss kokultivierter hUCB MNC auf Nekrose und Apoptose in neuralem Gewebe unter Sauerstoff-Glukose-Deprivation betrachtet. Die Studie ergab eine verbesserte funktionelle Rekonvaleszenz und eine geringere Ausprägung von Atrophie und Astroglianarbe bei Therapie innerhalb eines 72- Stunden-Zeitfensters. In vitro wurde eine signifikante Reduktion von Nekrose und Apoptose durch kokultivierte hUCB MNC beobachtet. Eine histologische Relokalisierung der intravenös applizierten Zellen war in keiner Therapiegruppe möglich. Die Integration der hUCB MNC ins Hirnparenchym stellt somit keine conditio sine qua non für die funktionelle Erholung nach Schlaganfall dar. Trotz des beobachteten erweiterten Zeitfensters ist die Translation dieses Therapieansatzes in die klinische Realität kritisch zu diskutieren, da weiterführende Studien unserer Arbeitsgruppe eine limitierte Wirksamkeit unter sehr praxisnahen Bedingungen (z.B. Einsatz kryokonservierter hUCB MNC) gezeigt haben. / Experimental treatment strategies using human umbilical cord blood mononuclear cells (hUCB MNCs) represent a promising option for alternative stroke therapies. An important point for clinical translation of such treatment approaches is knowledge on the therapeutic time window. Although expected to be wider than for thrombolysis, the exact time window for hUCB MNC therapy is not known. Our study aimed to determine the time window of intravenous hUCB MNC administration after middle cerebral artery occlusion (MCAO). Male spontaneously hypertensive rats underwent MCAO and were randomly assigned to hUCB MNC administration at 4h, 24h, 72h, 120h or 14d. Influence of cell treatment was observed by magnetic resonance imaging on days 1, 8 and 29 following MCAO and by assessment of functional neurological recovery. On day 30, brains were screened for glial scar development and presence of hUCB MNCs. Further, influence of hUCB MNCs on necrosis and apoptosis in post-ischemic neural tissue was investigated in hippocampal slices cultures. Transplantation within a 72h time window resulted in an early improvement of functional recovery, paralleled by a reduction of brain atrophy and diminished glial scarring. Cell transplantation 120h post MCAO only induced minor functional recovery without changes in the brain atrophy rate and glial reactivity. Later transplantation (14d) did not show any benefit. No evidence for intracerebrally localized hUCB MNCs was found in any treatment group. In vitro hUCB MNCs were able to significantly reduce post-ischemic neural necrosis and apoptosis. Our results for the first time indicate a time window of therapeutic hUCB MNC application of at least 72 hours. The time window is limited, but wider than compared to conventional pharmacological approaches. The data furthermore confirms that differentiation and integration of administered cells is not a prerequisite for poststroke functional improvement and lesion size reduction.

Der ischämische Schlaganfall ist global eine der bedeutendsten Volkskrankheiten. Die derzeit verfügbaren kurativen Therapieoptionen werden vorrangig durch ein enges therapeutisches Zeitfenster limitiert. Ziel der aktuellen Schlaganfallforschung ist die Entwicklung von über dieses Zeitfenster hinaus wirksamen Therapien. Ein vielversprechender neuer Ansatz ist die experimentelle Behandlung mit humanen Nabelschnurblutzellen. Diese Arbeit erforscht das therapeutische Zeitfenster für die systemische Therapie des ischämischen Schlaganfalls mittels mononukleärer Nabelschnurblutzellen (hUCB MNC) in spontanhypertensiven Ratten nach permanentem Verschluss der Arteria cerebri media (pMCAO). Hierzu wurden die Therapiezeitpunkte 4, 24, 72, 120 Stunden und 14 Tage nach experimentellem Schlaganfall in einem komplexen Studiendesign inklusive neurofunktioneller Tests, magnetresonanztomographischer und immunhistochemischer Verfahren untersucht. In vitro wurde der Einfluss kokultivierter hUCB MNC auf Nekrose und Apoptose in neuralem Gewebe unter Sauerstoff-Glukose-Deprivation betrachtet. Die Studie ergab eine verbesserte funktionelle Rekonvaleszenz und eine geringere Ausprägung von Atrophie und Astroglianarbe bei Therapie innerhalb eines 72- Stunden-Zeitfensters. In vitro wurde eine signifikante Reduktion von Nekrose und Apoptose durch kokultivierte hUCB MNC beobachtet. Eine histologische Relokalisierung der intravenös applizierten Zellen war in keiner Therapiegruppe möglich. Die Integration der hUCB MNC ins Hirnparenchym stellt somit keine conditio sine qua non für die funktionelle Erholung nach Schlaganfall dar. Trotz des beobachteten erweiterten Zeitfensters ist die Translation dieses Therapieansatzes in die klinische Realität kritisch zu diskutieren, da weiterführende Studien unserer Arbeitsgruppe eine limitierte Wirksamkeit unter sehr praxisnahen Bedingungen (z.B. Einsatz kryokonservierter hUCB MNC) gezeigt haben. / Experimental treatment strategies using human umbilical cord blood mononuclear cells (hUCB MNCs) represent a promising option for alternative stroke therapies. An important point for clinical translation of such treatment approaches is knowledge on the therapeutic time window. Although expected to be wider than for thrombolysis, the exact time window for hUCB MNC therapy is not known. Our study aimed to determine the time window of intravenous hUCB MNC administration after middle cerebral artery occlusion (MCAO). Male spontaneously hypertensive rats underwent MCAO and were randomly assigned to hUCB MNC administration at 4h, 24h, 72h, 120h or 14d. Influence of cell treatment was observed by magnetic resonance imaging on days 1, 8 and 29 following MCAO and by assessment of functional neurological recovery. On day 30, brains were screened for glial scar development and presence of hUCB MNCs. Further, influence of hUCB MNCs on necrosis and apoptosis in post-ischemic neural tissue was investigated in hippocampal slices cultures. Transplantation within a 72h time window resulted in an early improvement of functional recovery, paralleled by a reduction of brain atrophy and diminished glial scarring. Cell transplantation 120h post MCAO only induced minor functional recovery without changes in the brain atrophy rate and glial reactivity. Later transplantation (14d) did not show any benefit. No evidence for intracerebrally localized hUCB MNCs was found in any treatment group. In vitro hUCB MNCs were able to significantly reduce post-ischemic neural necrosis and apoptosis. Our results for the first time indicate a time window of therapeutic hUCB MNC application of at least 72 hours. The time window is limited, but wider than compared to conventional pharmacological approaches. The data furthermore confirms that differentiation and integration of administered cells is not a prerequisite for poststroke functional improvement and lesion size reduction.