One of the central questions in neuroscience is how the nervous system generates and regulates behavior. To understand the neural code for any behavior, an ideal experiment would entail (i) quantitatively defining that behavior, (ii) recording neuronal activity in relevant brain regions to identify the underlying neuronal circuits and eventually (iii) manipulating them to test their function. Novel methods in neuroscience have greatly advanced our abilities to conduct such experiments but are still insufficient. In this thesis, I developed methods for these three goals. In Chapter 2, I describe an automatic behavior identification and classification method for the cnidarian Hydra vulgaris using machine learning. In Chapter 3, I describe a fast volumetric two-photon microscope with dual-color laser excitation that can image in 3D the activity of populations of neurons from visual cortex of awake mice. In Chapter 4, I present a machine learning method that identifies cortical ensembles and pattern completion neurons in mouse visual cortex, using two-photon calcium imaging data. These methods advance current technologies, providing opportunities for new discoveries.

O envelhecimento é caracterizado por um progressivo declínio na função neuronal envolvendo tanto o sistema nervoso central como o periférico. O aumento da idade é verificado por alterações no número e no tamanho dos neurônios. Contudo, estes dados são controversos e pouco conhecidos nos gânglios periféricos. Desta forma, o presente estudo teve como objetivo estudar o gânglio mesentérico caudal (GMC) de cães em dois períodos distintos do desenvolvimento (maturação e envelhecimento) a procura de alterações microestruturais no gânglio e alterações quantitativas e morfométricas nos neurônios presentes neste. A importância do GMC está ligada à inervação simpática do intestino grosso, esfíncter anal interno e parcialmente do aparelho urogenital. Para o estudo foram utilizados nove cães domésticos, sem raça definida e machos, provenientes do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Estes animais foram divididos em três grupos etários com idades bem definidas (1-2 meses, 1-3 anos e 5-10 anos). Os gânglios foram processados para o estudo da microscopia de luz e as análises morfométrica e estereológica foram, respectivamente, realizadas utilizando o software de análise morfométrica KS 400 ZEISS® e o método estereológico do disector físico. O aumento da idade foi caracterizado por uma maior quantidade de tecido não neuronal entre os neurônios e um aumento no volume do gânglio. O tamanho dos neurônios, expresso pela sua área seccional, também aumentou com a progressão da idade, encontrando nos animais mais velhos a presença de grânulos de lipofucsina no interior destes. A relação núcleo-citoplasma dos neurônios do GMC tende a diminuir com o envelhecimento. Quanto às alterações quantitativas, o número total de neurônios no GMC aumenta com a idade em cães adultos e senis, diferentemente da densidade neuronal (neurônios/mm3) que diminui com a progressão da idade. Através do peso corpóreo do cão foi possível predizer os valores das diferentes variáveis estereológicas verificadas neste trabalho, sendo os valores obtidos muito próximos aos reais. / The aging is characterized by a progressive decline of neuronal function that involves both the central and the peripheral nervous system. Aging process is accompanied by changes in the number and size of neurons. However, these data are controversial and poorly known in the peripheral ganglia. In this way, the present investigation aimed to study the dog\'s caudal mesenteric ganglion (CMG) in different phases of aging, looking for qualitative, quantitative and morphometric alterations in these neurons. The importance of the CMG is associated with the innervation of the lower large intestine, internal anal sphincter and partially the urogenital system. To the study, was used nine males, mongrel, domestic dogs from the Veterinary Hospital of the Veterinary and Zoöthecny College of the São Paulo University. They were divided into three different well defined aged groups (1-2 months old; 1-3 years old; 5-10 years old). The ganglia were processed for the light microscopy study. The morphometric and stereological analyses were, respectively, done using morphometric analyses software KS 400 Zeiss® and the stereological physical disector method. The increase of age was caractherized by a major amount of the non-neuronal tissue between the neurons and a increase of ganglion volume. The size of the neurons, represented by their sectional area, also increased with the age, being frequent, the presence of lipofucsin granules in old animals. The nucleus-citoplasmic relation of neurons of the CMG tend to decrease with the age. Referring to the quantitative changes, the total number of neurons in the CMG increased by aging in adults and elder dogs, differently of the neuronal density (neurons/mm3) which decreased with the aging. Through the body weight of the dog, it was possible to predict the values of the different stereological variables represented in this paper, where, the obtained values were close to the normal rates.

Circadian rhythms refer to the physiological and biological processes that fluctuate over a 24-hour period. These rhythms are found in most living things such as animals, plants and fungi. In mammals, circadian rhythms are mainly generated and regulated by the suprachiasmatic nucleus (SCN).The period (Per1) gene in the SCN plays a key role in directing circadian rhythms. Per1 expression increases during the day and decreases at night. The neurons which express the Per1 gene show different behaviours to non-Per1 neurons during the day-night cycle. Per1 neurons are in a state of repetitive firing in the morning while in the late morning they display a bursting behaviour. In the afternoon, Per1 neurons divide into two groups: GA and GB, where GA is in a repetitive firing state and GB is silent. At night, these neurons are generally in a quiescent state but late at night they generate spikes. In this study, a standard Hodgkin-Huxley type model was used to study the behaviours of Per1 neurons over the day-night cycle using bifurcation analysis. In this model, the potassium and calcium currents carry the circadian rhythms which are modelled by their conductance. The currents had a significant impact on Per1 neuron behaviours. Furthermore, by changing some of the model parameters, different bistability mechanisms were examined. The study was extended to explore the effect of noise from other neurons on Per1 neuron behaviours. It was shown that noise plays a crucial role in inducing some of the Per1 neuron behaviours and that some Per1 neuron behaviours are fully induced by this noise, e.g. the late morning and late night behaviours. In contrast, the noise was found not to have any significant effect on other Per1 neuron behaviours other than the two behaviours that are observed in the afternoon.

Dans le domaine médical, une meilleure connaissance de la fonctionmotrice est susceptible de permettre d’établir des thérapies adaptéesà chaque lésion motrice et des outils d’études et de dépistage dansle cas de maladies neurodégénératives. Dans le domaine de la réalitévirtuelle, la reconnaissance du mouvement est un point angulaire dansl’interaction de l’avatar ou de la personne en immersion avec sonenvironnement. Plusieurs travaux ont été menés dans le but de proposerdes approches de classification du mouvement humain. L’idée principalede ces méthodes est d’extraire des invariants des données enregistréesafin de les regrouper en clusters. Cependant, l'étude du mouvementhumain avec des systèmes de capture de mouvement génère une quantitéde données volumineuse, avec des relations non linéaires entre elles.Les méthodes présentées dans la littérature scientifique utilisent cesdonnées soit directement comme entrée à des algorithmes declassification, soit en appliquant une méthode de réductiondimensionnelle, comme l’analyse en composantes principales avant laclassification. Ces méthodes restent extrêmement sensibles au bruitblanc pendant l'enregistrement ainsi qu’aux différences morphologiquesentre les sujets. Dans notre travail, nous allons présenter uneméthodologie de classification et de reconnaissance du mouvementhumain, qui se base sur l’analyse topologique des donnéescinématiques. L’analyse topologique sera réalisée via la persistancehomologique, qui est une méthode d’analyse des données volumineusesqui permet de leur associer une signature topologique. On combineracette méthode d’analyse topologique avec des algorithmesd’apprentissage afin d’augmenter la précision de la reconnaissance desmouvements en réduisant l'impact des différences morphologiques entreles sujets ainsi que l’impact du bruit blanc issu pendant l'étaped’acquisition du mouvement. Par ailleurs, on combinera la méthoded’analyse topologique avec un algorithme de réseaux de neuronestemporels, afin de construire une approche qui permet de prédire lasuite d’un mouvement à partir d’une partie d’un intervalled’enregistrement.Les résultats ont montré la capacité de l’approche proposée à obtenirune précision avec un taux élevé lors de la classification, ainsi quesa robustesse face au bruit blanc et aux différences morphologiquesentre les personnes. Les résultats ont montré aussi le cout élevé entemps de calcul de notre approche. Nous avons proposé des méthodes(algorithme et parallélisme) de façon à réduire les temps de calculs. / In the medical field, a better knowledge of the motor function isimportant for us to determine therapies adapted to each motor lesion andtools of studies and screening for neurodegenerative diseases. In thedomain of virtual reality, motion recognition is an issue in theinteraction of the avatar or the user in immersion with theirenvironment.Several studies have been conducted with the aim of proposingapproaches to the classification of human movement. The main idea ofthese methods is to extract invariants from the recorded data in orderto order them into clusters. However, the study of human motion withmotion capture systems generates a big quantity of data with nonlinearrelations between them. The presented methods in the scientificliterature use these data either directly as input to classificationalgorithms or by applying a dimensional reduction method such asprincipal component analysis prior to classification. These methodsremain extremely sensitive to white noise during recording as well asmorphological differences between subjects.In our work, we will present a methodology of classification andrecognition of human movement which is based on the topologicalanalysis of kinematic data. Topological analysis will be performed viahomological persistence which is a large data analysis method thatallows them to be topologically signed. This method of topologicalanalysis will be combined with learning algorithms to increase theaccuracy of motion recognition by reducing the impact of morphologicaldifferences between subjects, as well as the impact of white noiseissued during the step of movement acquisition. Also, we will combinethe topological analysis method with a temporal neural networkalgorithm in order to build an approach that allows to predict thecontinuation of a movement from a part of a recording interval.The results showed the ability of the proposed approach to achievehigh accuracy at classification, as well as its robustness againstwhite noise and morphological differences between subjects. Theresults also showed the high cost in computing time of our approachwhich we tried to reduce by modifying its steps and by rewriting thecode so that it can be executed in parallel.

Yin Xiao-Lu. / "February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Gemin5 是運動神經元綜合體(SMN Complex)的其中一個組件，這綜合體的主要功能是控制小型胞核核糖核蛋白(UsnRNPs)的合成。這些小型胞核核糖核蛋白組合成剪接核糖核酸前體(pre-mRNA)的剪接體(Spliceosome)，使核糖核酸分子可以用來翻譯成蛋白質。失去運動神經元綜合體功能引致脊髓肌肉萎縮症。果蠅是其中一個用作研究人類疾病重要的生物。更重要的是，部分組成運動神經元綜合體的組件也存在於果蠅。是次研究是利用遺傳方式在果蠅內研究Gemin5 的同源基因-- rigor mortis (rig) 的作用。果蠅帶有rig 突變基因表現神經肌肉接頭(neuromuscular junction)上的缺陷和異常的運動行為。這表明，rig 的功能可能與神經退化性疾病有關。為了進一步了解rig 的功能途徑(functional pathway)，已進行了一個利用移除突變體的基因過濾實驗，研究鎖定了 12 個染色體部份可能和rig 有基因上的相互作用，進一步研究與rig 有相互作用的基因有助於了解rig 的功能及研究脊髓肌肉萎縮症的發病機制。 / Gemin5 is a component of the Survival of Motor Neuron (SMN) complex, which is a protein complex regulating biogenesis of various Uridine-enriched small nuclear ribonucleoproteins (UsnRNPs). These UsnRNPs form the molecular machinery spliceosome, which mediates pre-messenger RNA splicing, an important mechanism before an mRNA molecule can be used to translate proteins. Loss-of-function of the SMN complex is now known to cause the neurodegenerative disease, Spinal Muscular Atrophy. Drosophila is one of the well-characterized model organisms for studying human diseases. More importantly, components of the SMN complex are also found in Drosophila. Here, I studied the function of rigor mortis (rig), the Gemin5 orthologue, in Drosophila using a genetic approach. Drosophila carrying mutations in the rig gene show defects in the neuromuscular junction (NMJ) and display abnormal motor behavior. This suggests that the function of rig may link to the neurodegenerative disease. To further characterize the function of rig, a genetic screen was carried out. Twelve chromosomal regions encoding possible rig-interacting genes were identified. Further characterization of these rig-interacting genes may help us better understand the function of rig. / Detailed summary in vernacular field only. / Cheng, Yat Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-125). / Abstracts also in Chinese. / ABSTRACT --- p.i / ABSTRACT IN CHINESE --- p.ii / ACKNOWLEDGEMENT --- p.iii / LIST OF ABBREVIATIONS --- p.iv / LIST OF FIGURES --- p.v / LIST OF TABLES --- p.vi / TABLE OF CONTENTS --- p.vii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Introduction of rigor mortis / Chapter 1.1.1 --- Orthologue of Gemin5 in Drosophila --- p.1 / Chapter 1.1.2 --- Published Phenotypic Analyses of rig Mutants --- p.1 / Chapter 1.2 --- Introduction of Gemin5 / Chapter 1.2.1 --- Introduction of Gemins --- p.4 / Chapter 1.2.2 --- Structural Properties of Gemin5 --- p.4 / Chapter 1.2.3 --- Gemin5-interacting partners --- p.7 / Chapter 1.2.4 --- Gemin5 as a Component of the Survival of Motor Neuron (SMN) Complex --- p.7 / Chapter 1.2.5 --- Function of the SMN Complex and Spinal Muscular Atrophy --- p.11 / Chapter 1.3 --- Drosophila as a Model Organism / Chapter 1.3.1 --- Advantages of Using Drosophila as a Model Organism --- p.11 / Chapter 1.3.2 --- Neuromuscular Junction of Drosophila --- p.15 / Chapter 1.4 --- Aim of the Present Study --- p.19 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila Culture / Chapter 2.1.1 --- Culture Medium --- p.20 / Chapter 2.1.2 --- Drosophila Stocks and Crosses Maintenance --- p.20 / Chapter 2.1.3 --- Larvae Collection --- p.21 / Chapter 2.1.3.1 --- Reagents --- p.21 / Chapter 2.1.3.2 --- Procedures --- p.21 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Reagents --- p.23 / Chapter 2.2.2 --- Drosophila S2R⁺ Cell Culture --- p.24 / Chapter 2.2.3 --- Establishment of Stable S2R⁺ Cells --- p.24 / Chapter 2.3 --- Genomic Polymerase Chain Reaction (PCR) / Chapter 2.3.1 --- Reagents --- p.25 / Chapter 2.3.2 --- Genomic DNA Extraction from a Single Larva --- p.26 / Chapter 2.3.3 --- Primer Design --- p.26 / Chapter 2.3.4 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.4 --- Behavioral Assay / Chapter 2.4.1 --- Stable S2R⁺ Cell Staining --- p.29 / Chapter 2.4.1.1 --- Reagents --- p.29 / Chapter 2.4.1.2 --- Procedures --- p.30 / Chapter 2.4.2 --- Larvae Staining --- p.31 / Chapter 2.4.2.1 --- Reagents --- p.31 / Chapter 2.4.2.2 --- Larvae Dissection --- p.32 / Chapter 2.4.2.3 --- Larval Muscle Staining --- p.33 / Chapter 2.4.2.4 --- Larval Neuromuscular Junction Staining --- p.33 / Chapter 2.5 --- Microscopy / Chapter 2.5.1 --- Light Microscopy --- p.34 / Chapter 2.5.1.1 --- Microscopic Observation of Larval Movement --- p.34 / Chapter 2.5.1.2 --- Quantification of Larval Contraction Rate --- p.34 / Chapter 2.5.1.3 --- Quantification of Larval Travelling Distance --- p.34 / Chapter 2.5.2 --- Fluorescence Microscopy --- p.35 / Chapter 2.5.2.1 --- Microscopic Observation of Larval Muscle --- p.35 / Chapter 2.5.2.2 --- Microscopic Observation of Stable S2R⁺ Cells --- p.35 / Chapter 2.5.3 --- Confocal Microscopy --- p.36 / Chapter 2.5.3.1 --- Microscopic Observation of Larval Neuromuscular Junction --- p.36 / Chapter 2.5.3.2 --- Quantification of Larval Neuromuscular Junction --- p.36 / Chapter 2.6 --- Generation of transgenic fly lines expressing rig transgene / Chapter 2.6.1 --- Polymerase Chain Reaction --- p.36 / Chapter 2.6.2 --- Agarose Gel Electrophoresis --- p.38 / Chapter 2.6.2.1 --- Reagents --- p.38 / Chapter 2.6.2.2 --- Procedures --- p.39 / Chapter 2.6.3 --- Restriction Digestion --- p.39 / Chapter 2.6.4 --- Ligation Reaction --- p.39 / Chapter 2.6.5 --- Bacterial Transformation --- p.40 / Chapter 2.6.5.1 --- Reagents --- p.40 / Chapter 2.6.5.2 --- Procedures --- p.40 / Chapter 2.6.6 --- Bacterial Glycerol Stock for Long-term Storage --- p.41 / Chapter 2.7 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting / Chapter 2.7.1 --- Reagents --- p.41 / Chapter 2.7.2 --- Lysate Preparation of Stable S2R⁺ Cells, Adult Fly Heads and Larvae --- p.44 / Chapter 2.7.2.1 --- Stable S2R+ Cells --- p.44 / Chapter 2.7.2.2 --- Adult Fly Heads --- p.44 / Chapter 2.7.2.3 --- Larvae --- p.45 / Chapter 2.7.3 --- SDS-Polyacrylamide Gel Electrophoresis --- p.45 / Chapter 2.7.4 --- Immunoblotting --- p.45 / Chapter CHAPTER 3. --- PHENOTYPIC CHARACTERIZATION OF RIG MUTANT / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Re-balancing of rig Mutant Fly Lines Over the Cy; Tb Compound Balancer for Genotype Identification --- p.48 / Chapter 3.3 --- Verification of Model Genotype --- p.49 / Chapter 3.4 --- rig Mutant Larvae Displayed Abnormal Motor Behavior / Chapter 3.4.1 --- Contraction Rate of rig Mutant Larvae --- p.54 / Chapter 3.4.2 --- Traveling Distance of rig Mutant Larvae --- p.56 / Chapter 3.5 --- rig Mutant Larvae Showed Normal Body Wall Musculature --- p.58 / Chapter 3.6 --- rig Mutant Larvae Displayed Defects in the Neuromuscular Junction / Chapter 3.6.1 --- rig Mutant Larvae Showed Branching Defects --- p.60 / Chapter 3.6.2 --- rig Mutant Larvae Showed Fewer Boutons Number --- p.62 / Chapter 3.7 --- rig Mutant Larvae Showed Normal Active Zone Pattern --- p.64 / Chapter 3.8 --- Discussion --- p.66 / Chapter CHAPTER 4. --- A GENETIC SCREEN TO IDENTIFY GENES THAT INTERACT GENETICALLY WITH RIG / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Candidates and Design of the Screen --- p.72 / Chapter 4.3 --- Re-balancing of Deletion Lines Over the Cy; Tb Compound Balancer --- p.75 / Chapter 4.4 --- Identification of Chromosomal Regions That Genetically Interact With rig --- p.75 / Chapter 4.5 --- Identification of NMJ Genes That Genetically Interact With rig --- p.80 / Chapter 4.6 --- Discussion --- p.83 / Chapter CHAPTER 5. --- ATTEMPTS TO INVESTIGATE RIG FUNCTION IN PRE-AND POST-SYNAPTIC REGIONS OF THE NMJ / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.2 --- Transgenic Rescue Experiment by Transgenic Expression of rig in rig Mutant / Chapter 5.2.1 --- Design of the Rescue Experiment --- p.90 / Chapter 5.2.2 --- Construct of pUAST-rig-FLAG --- p.93 / Chapter 5.2.3 --- Construct of the pUAST-myc-rig --- p.98 / Chapter 5.3 --- Tissue Specific Knockdown of rig expression --- p.102 / Chapter 5.4 --- Discussion --- p.105 / Chapter CHAPTER 6. --- ESTABLISHMENT OF AN INDUCIBLE S2R⁺ CELL MODEL FOR RIG OVEREXPRESSION / Chapter 6.1 --- Introduction --- p.108 / Chapter 6.2 --- Detection of Rig Protein in S2R⁺ Cells by Immunoblotting --- p.111 / Chapter 6.3 --- Detection of Rig Protein in S2R⁺ Cells by Immunostaining --- p.111 / Chapter 6.4 --- Detection of RNA in Immunopurified Rig Protein --- p.113 / Chapter 6.5 --- Discussion --- p.115 / Chapter CHAPTER 7. --- GENERAL DISCUSSION --- p.117 / References --- p.120 / Appendices --- p.126

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset paralytic disorder for which there is currently no cure. Underlying the disease mechanism of ALS is the spontaneous pathologic degeneration of motor neurons (MNs). Understanding the molecular mechanisms underlying spontaneous and selective MN demise is critical to the development of rational therapeutic strategies. In the current work, utilizing established in vitro models of ALS, I demonstrate that necroptosis, a form of caspase-independent programmed cell death (PCD), drives MN death. Pharmacologic inhibition and/or genetic silencing of receptor interacting protein kinase-1 (RIPK1), receptor interacting protein kinase-3 (RIPK3), and mixed lineage kinase domain-like-protein (MLKL) rescued MN death in vitro. While this core machinery was conserved, the requirement of nuclear factor kappa-B (NF-κB) and Bcl-2-associated X protein (Bax) deviated from known models of necroptosis. This divergence led me to consider that there may be a MN-specific program of necroptosis. Thus, I then used unbiased approaches, by meta-analyzing a gene expression signature captured from MNs undergoing cell death in vitro, to explore MN cell death drivers that may be engaged upstream or downstream to RIPK1/RIPK3/MLKL. I also explored the relevance of necroptosis to MN disease in vivo, in part by deleting RIPK3 from a genetic mouse model of familial ALS. Overall this approach did not rescue motor neuron loss, and there was no improvement in motor function, disease onset, or survival in these animals. I conclude that while necroptosis machinery drives motor neuron death in in vitro models of ALS, more work needs to be done to (1) assess the motor neuron-specific cell death program, and (2) evaluate the relationship, if any, of necroptosis to motor neuron disease in vivo.

The primary motor cortex (M1) is a key brain area for the generation and control of motor behaviour. Output from M1 can be driven in part by long-range inputs from a collection of thalamic nuclei termed the motor thalamus (MTh), but how MTh input shapes activity in M1 and forelimb motor behaviour remains largely unresolved. To address this issue, we first defined the 3D anatomical coordinates of mouse forelimb motor thalamus (MThFL) by employing conventional retrograde and virus-based tracing methods targeted to the forelimb region of M1 (M1FL). These complimentary approaches defined MThFL as a ~0.8 mm wide cluster of neurons with anatomical coordinates 1.1 mm caudal, 0.9 mm lateral to bregma and 3.2 mm below the pial surface. Thus, MThFL incorporates defined areas of the ventrolateral, ventral anterior and anteromedial thalamic nuclei. To investigate the importance of M1FL and MThFL during skilled motor behaviour, we developed and optimised a quantitative behavioural paradigm in which head-restrained mice execute forelimb lever pushes in response to an auditory cue to receive a water reward. Forelimb movement trajectories were mapped using high-speed digital imaging and multi-point kinematic analysis. We inactivated both M1FL and MThFL of mice performing this motor behaviour using a pharmacological strategy, which in both cases resulted in a significant reduction in task performance. Inactivating M1FL significantly affected forelimb coordination and dexterity, resulting in erratic motion and posture. In contrast, mice with MThFL inactivated displayed a reduction in total motor output, although correct posture was maintained. We performed extracellular recordings in MThFL of expert-level mice, demonstrating that motor thalamic output during execution of task was dominated by a robust response to the onset of the auditory cue. Cue-evoked responses were also observed in motor thalamic neurons of naive mice. We have developed a novel solution to the stability problem encountered when performing whole-cell patch-clamp recordings from the motor cortex of head-restrained mice performing forelimb motor behaviour, and present preliminary recordings maintained through the execution of forelimb behaviour.

The balance between excitatory and inhibitory synaptic transmission within and across neurons in active networks is crucial for cortical function and may allow for rapid transitions between stable network states. GABAergic interneurons mediate the majority of inhibitory transmission in the cortex, and therefore contribute to the global balance of activity in neuronal networks. Disruption in the network balance due to impaired inhibition has been implicated in several neuropsychiatric diseases (Marin 2012). Both schizophrenia and autism are two highly heritable cognitive disorders with complex genetic aetiologies but overlapping behavioural phenotypes that share common imbalances in neuronal network activity (Gao & Penzes 2015). An increasing body of evidence suggests that functional abnormalities in a particular group of cortical GABAergic interneurons expressing the calcium-binding protein parvalbumin (PV) are involved in the pathology of these disorders (Marin 2012). As deficits in this neuronal population have been linked to these disorders it could be useful to target them and increase their activity. A conserved feature in PV cells is their unusually low input resistance compared to other neuronal populations. This feature is regulated by the expression of leak K+ channels, believed to be mediated in part by TASK and TREK subfamily two-pore K+ channels (Goldberg et al. 2011). The selective blockade of specific leak K+ channels could therefore be applied to increase the activity of PV cells. In this thesis, specific TASK-1/3 and TREK-1 channel blockers were applied in cortical mouse slices in an attempt to increase the output of PV cells. The blockade of either channel did not successfully increase the amplitude of PV cell-evoked inhibitory postsynaptic currents (IPSCs) onto principal cells. However, while the blockade of TASK-1/3 channels failed to depolarise the membrane or alter the input resistance, the blockade of TREK-1 channels resulted in a small but significant depolarisation of the membrane potential in PV cells. Interestingly, TREK-1 channel blockade also increased action potential firing of PV cells in response to given current stimuli, suggesting that TREK-1 could be a useful target for PV cell modulation. These results demonstrate for the first time the functional effects of using specific two-pore K+ channel blockers in PV cells. Furthermore, these data provide electrophysiological evidence against the functional expression of TASK-1/3 in PV cells. It could therefore be interesting to further characterise the precise subtypes of leak K+ channels responsible for their low resistivity. This would help to classify the key contributors of the background K+ conductances present in PV cells in addition to finding suitable targets to increase their activity.

One central question of neuroscience asks how a neural system can generate the diversity of complex behaviors needed to meet the range of possible demands placed on an organism by an ever changing environment. In many cases, it appears that animals assemble complex behaviors by recombining sets of simpler behaviors known as behavioral primitives. The crawling behavior of the nematode worm Caenorhabditis elegans represents a classic example of such an approach since worms use the simple behaviors of forward and reverse locomotion to assemble more complex behaviors such as search and escape. The relative simplicity and well-described anatomy of the worm nervous system combined with a high degree of genetic tractability make C. elegans an attractive organism with which to study the neural circuits responsible for assembling behavioral primitives into complex behaviors. Unfortunately, difficulty probing the physiological properties of central synapses in C. elegans has left this opportunity largely unfulfilled. In this dissertation we address this challenge by developing techniques that combine whole-cell patch clamp recordings with optical stimulation of neurons. We do this using transgenic worms that express the light-sensitive ion channel Channelrhodopsin-2 (ChR2) in putative pre-synaptic neurons and fluorescent protein reporters in the post-synaptic neurons to be targeted by electrodes. We first apply this new approach to probe C. elegans circuitry in chapter II where we test for connectivity between nociceptive neurons known as ASH required for sensing aversive stimuli, and premotor neurons required for generating backward locomotion, known as AVA. In chapter III we extend our analysis of the C. elegans locomotory circuit to the premotor neurons required for generating forward locomotion, known as AVB. We identify inhibitory synaptic connectivity between ASH and AVB and between the two types of premotor neurons, AVA and AVB. Finally, we use our observations to develop a biophysical model of the locomotory circuit in which switching emerges from the attractor dynamics of the network. Primitive selection in C. elegans may thus represent an accessible system to test kinetic theories of decision making. This dissertation includes previously published co-authored material.