Maternal alcohol abuse during pregnancy can result in a range of structural and functional abnormalities that include lifelong physical, mental, behavioral and learning disabilities, now collectively termed as Fetal Alcohol Spectrum Disorders (FASD). The incidence of FASD is now estimated be as high as 10 per 1000 live births. Each year, 40,000 babies are born with FASD in the United States at an estimated cost of $1.4 million per individual and total cost of $6 billion. Because of the magnitude of this problem and because the incidence has not decreased in spite intensive efforts to educate women to not abuse alcohol during pregnancy, ways to prevent or mitigate the effects of prenatal alcohol exposure must be explored in addition to education. Therefore, we wished to identify the precise mechanisms by which alcohol mediates the neurodevelopmental damage in order to develop intervention/amelioration strategies. The present study was conducted using an ovine model system. The large body mass of the ovine fetus, the longer gestation that is more similar to that of humans, and that all three trimester equivalents occur in utero, make the sheep an excellent model to study the effects of alcohol on the developing fetus. Our study establishes that maternal alcohol exposure does not result in fetal cerebral hypoxia. Instead, alcohol results in hypercapnea and acidemia leading to a cascade of events in the maternal and fetal compartments that include deficits in the levels of glutamine and glutamine-related amino acids, alterations in endocrine axes, oxidative stress, alteration in cardiovascular homeostasis and fetal neuronal loss. Further, we demonstrate that inhibiting the novel two-pore domain acid sensitive potassium channel (TASK) expressed in the cerebellar granule cells and the peripheral and central chemoreceptors may prove to a be potential therapeutic strategy. Preventive strategies that are safe to use in pregnant women and that involve glutamine-related pathways are also suggested. Finally, the study also establishes the beneficial effects of moderate alcohol consumption on the fetal skeletal system.

Mutations of the á1A subunit of P/Q-type voltage-gated calcium channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6. These disorders include phenotypes such as a progressive cerebellar atrophy and ataxia. The leaner mouse also carries a mutation in the alpha(1A) subunit of P/Q-type voltage-gated calcium channels, which results in a severe cerebellar atrophy and ataxia. The leaner mutation causes reduced calcium ion influx upon activation of P/Q-type voltage-gated calcium channels. This disrupts calcium homeostasis and leads to a loss of cerebellar neurons, including cerebellar Purkinje cells. Because of its similarities with human P/Qtype voltage-gated calcium channel mutations, leaner mouse has served as a model for these disorders to aid our understanding of calcium channel function and neurodegeneration associated with calcium channel dysfunction. The aims of this dissertation were: (1) to precisely define the timing and spatial pattern of leaner Purkinje cell death and (2) to assess the role of caspases and specifically of caspase 3 in directing leaner Purkinje cell death. We used the mechanism independent marker for cell death Fluoro-Jade and demonstrated the leaner Purkinje cell death begins around postnatal day 25 and peaks at postnatal day 40 to 50. Based on this temporal pattern of Purkinje cell death we then investigated the role of caspases in leaner Purkinje cell death. These studies showed that caspase 3 is specifically activated in dying leaner cerebellar Purkinje cells. In addition, in vitro inhibition of caspase 3 activity partially rescued leaner Purkinje cells. Further investigation revealed that caspase 3 activation may be working together with or in response to macroautophagy. This study also indicated a potential role for mitochondrial signaling, demonstrated by the loss of mitochondrial membrane potential in leaner cerebellar Purkinje cells. However, our study revealed that if the loss of mitochondrial membrane potential is associated with leaner Purkinje cell death, this process is not mediated by the mitochondrial protein cytochrome C.

Short-term facilitation, a form of synaptic plasticity, takes place at GABAergic synapses between cerebellar Purkinje cells (PCs). We studied the mechanism of this short-term facilitation by directly patch-clamp recording from a PC axon terminal in cerebellar cultures. We show that the Ca2+ currents elicited by high-frequency action potentials were augmented depending on intracellular [Ca2+] on the terminal. The facilitation of synaptic transmission showed 4-5th power dependence on the Ca2+ current facilitation, and was abolished when the Ca2+ current facilitation was supressed. We conclude that short-term facilitation of Ca2+ currents predominantly mediates short-term facilitation at synapses between PCs. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

La cellule de Purkinje (PC) est l'élément central du réseau neuronal du cortex cérébelleux et possède un arbre dendritique très développé qui se développe au cours des trois premières semaines post-natales chez la souris. Cette arborisation nécessite de nombreux réarrangement du cytosquelette, un processus contrôlé par les GTPases et leurs régulateurs, les GEFs et les GAPs, dans de nombreux types cellulaires. Au cours de ma thèse, j'ai étudié l'implication de la signalisation des RhoGTPases dans le développement post-natal du cervelet, et plus particulièrement des PCs chez la souris. Afin d'identifier de nouveaux acteurs de la signalisation des RhoGTPases impliqués dans la différenciation des PCs, nous avons établi le profil d'expression de toutes les GTPases et des GEFs de la famille DOCK à différents stades de développement de ces cellules (P3, P7, P15, P20) par Q-PCR en temps réel. Cette approche globale nous a permis d'identifier une GTPase, RhoQ, et un GEF, DOCK10, dont l'expression est très fortement augmentée au cours du développement des PCs. Nous avons montré que l'extinction de leur expression par infection lentivirale dans un modèle de coupes organotypiques de cervelet ou dans des neurones d'hippocampe entraine une très forte diminution du nombre d'épines dendritiques, révélant un rôle crucial de ces protéines dans la différenciation des PCs. / Purkinje cell (PC) occupy a central and integrative position in the synaptic network of the cerebellum and have the most elaborate dendritic tree among CNS neurons, which develops remarkably in the first three postnatal weeks in mice. This arborization requires intensive actin cytoskeleton remodeling, a process known in many cell types to be controlled by Rho GTPases and their regulators, GEFs and GAPs. During my thesis, I investigated the importance of Rho signaling during postnatal mouse cerebellar development, focusing on PC differentiation.In order to identify novel regulators of PC differentiation among members of the Rho signaling pathway, I undertook a global approach, comparing gene expression profiles of all mammalian Rho GTPases and all GEFs of the DOCK family at various stages of postnatal PC differentiation (P3, P7, P15 and P20) using real-time quantitative PCR. My global approach has allowed the identification of two Rho signaling actors, the GTPase RhoQ and the RhoGEF DOCK10, whose expressions increase dramatically during cerebellar development. Lentiviral shRNA-mediated knock down of their expression in organotypic cerebellar cultures and in hippocampal neurons showed strong dendritic spine defects, revealing an essential role for these proteins in PC differentiation.

T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.

Running title: Climbing fibers in myosin Va mutants

T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.

Neurons can encode information using the rate of their action potentials, making the relation between input rate and output rate a prominent feature of neuronal information processing. This relation, known as I{O function, can rapidly change in response to various factors or neuronal processes. Most noticeably, a neuron can undergo a multiplicative operation, resulting in a change of the slope of its I{O curve, also know as gain change. Gain changes represent multiplicative operations, and they are wide- spread. They have been found to play an important role in the encoding of spatial location and coordinate transformation, to signal amplification, and other neuronal functions. One of the factors found to introduce and control neuronal gain is synaptic Short Term Depression (STD). We use both integrate-and- re and conductance based neuron models to identify the effect of STD in excitatory and inhibitory modulatory input. More specifically, we are interested in the effect of STD at the inhibitory synapse from Purkinje cells to cerebellar nucleus neurons. Using a previously published, biologically realistic model, we find that the presence of STD results in a gain change. Most importantly we identify STD at the inhibitory synapse to enable excitation-mediated gain control. To isolate the mechanism that allows excitation to control gain, even though STD is applied at a different synapse, we first show that the overall effect is mediated by average conductance. Having done this, we find that the effect of STD is based on the non-linearity introduced in the relation between input rate and average conductance. We find this effect to vary, depending on the position of the I{O function on the input rate axis. Modulatory input shifts the I{O curve along the input rate axis, consequently shifting it to a position where STD has a different effect. The gain differences in the STD effects between the two positions enable excitation to perform gain control.

T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs. / Medicine, Faculty of / Graduate

Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be esential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. Teese data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.