The past three decades of synaptic research have provided new insights into synapse biology. While synapses are still considered the fundamental connectors between the nerve cells in the central nervous system, they are no longer seen as simple neuron-to-neuron contacts. In fact, the estimated 100 trillion of human synapses are extremely complex, diverse and capable of performing sophisticated computational operations giving rise to advanced repertoires of cognitive and organic behaviours. These intricate synaptic properties mean that existing methodologies for quantifying and characterising synapses are inadequate. Yet, understanding of synapse biology is crucial to deciphering human pathology as disruptions in synapse numbers, architecture and function have already been linked to many human brain disorders. The purpose of this PhD was to evaluate a novel, high-throughput synaptic protein quantification method at a single synapse resolution in human post-mortem brain tissue. The method has already been successfully tested in our laboratory in genetically engineered mice, whereby synapses have been systematically quantified across a large number of areas to generate the first molecular maps of synapses, the synaptome maps. In this project, methods have been developed to label human brain tissue with postsynaptic density protein 95 (PSD-95), the most common postsynaptic protein. We describe the use of PSD-95 combined with confocal microscopy and computational image analysis to quantify synaptic puncta immunofluorescence (IF) parameters in the human brain. In the first part of this study, the new method was used to quantify PSD-95 IF across selected 20 human brain regions to generate first PSD-95 human synaptome map. In the second part, PSD-95 IF was systematically assessed across 16 hippocampal subregions. Finally, we confirmed that our novel synaptic quantification method was sensitive to hippocampal synaptic losses in patients with Alzheimer's Disease (AD). Such a high degree of systematic synapse quantification has not previously been reported in human brain tissue. Our method is a promising approach for synaptic protein quantification in tissue with several potential applications in diagnosis and development of therapeutics for neurological and psychiatric disorders.

During nervous system development, thousands of distinct neuronal cell types are generated and assembled into highly precise circuits. The proper wiring of these circuits requires that developing neurons recognize their appropriate synaptic partners. Analysis of a vertebrate spinal circuit that controls motor behavior reveals distinct synaptic connections of two types of inhibitory interneurons, a ventral V1 class that synapses with motor neurons and a dorsal dI4 class that selectively synapses with proprioceptive sensory neuron terminals that are located on or in close proximity to motor neurons. What are the molecular and cellular programs that instruct this remarkable synaptic specificity? Are only subsets of these interneurons capable of integrating into this circuit, or do all neurons within the same class behave similarly? The ability to answer such questions, however, is hampered both by the complexity of the spinal cord, where many different neuronal cell types can be found synapsing in the same area; as well as by the challenge of obtaining enough neurons of a particular subtype for analysis. Meanwhile, pluripotent stem cells have emerged as powerful tools for studying neural development, particularly because they can be differentiated to produce large amounts of diverse neuronal populations. Mouse embryonic stem cell-derived neurons can thus be used in a simplified in vitro system to study the development of specific neuronal cell types as well the interactions between defined cell types in a controlled environment. Using stem cell-derived neurons, I investigated how the V1 and dI4 cardinal spinal classes differentiate into molecularly distinct subtypes and acquire cell type-specific functional properties, including synaptic connectivity. In Chapter Two, I describe the production of lineage-based reporter stem cell lines and optimized differentiation protocols for generating V1 and dI4 INs from mouse embryonic stem cells, including confirming that they have molecular and functional characteristics of their in vivo counterparts. In Chapter Three, I show that a well-known V1 interneuron subtype, the Renshaw cell, which mediates recurrent inhibition of motor neurons, can be efficiently generated from stem cell differentiation. Importantly, manipulation of the Notch signaling pathway in V1 progenitors impinges on V1 subtype differentiation and greatly enhances the generation of Renshaw cells. I further show that sustained retinoic acid signaling is critical for the specific development of the Renshaw cell subtype, suggesting that interneuron progenitor domain diversification may also be regulated by spatially-restricted cues during embryonic development. In Chapter Four, using a series of transplantation, rabies virus-based transsynaptic tracing, and optogenetics combined with whole-cell patch-clamp recording approaches, I demonstrate that stem cell-derived Renshaw cells exhibit significant differences in physiology and connectivity compared to other V1 subpopulations, suggesting that synaptic specificity of the Renshaw cell-motor neuron circuit can be modeled and studied in a simplified in vitro co-culture preparation. Finally, in Chapter Five, I describe ongoing investigations into molecular mechanisms of dI4 interneuron subtype diversification, as well as approaches to studying their synaptic specificity with proprioceptive sensory neurons. Overall, my results suggest that our stem cell-cell based system is well-positioned to probe the functional diversity of molecularly-defined cell types. This work represents a novel use of embryonic stem cell-derived neurons for studying inhibitory spinal circuit assembly and will contribute to further understanding of neural circuit formation and function during normal development and potentially in diseased states.

Thèse de doctorat : Neurosciences : Strasbourg 1 : 2008. / Extrait en partie de périodique. Titre provenant de l'écran-titre. Bibliogr. p. 147-161.

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie. Neurosciences : Strasbourg 1 : 2006. / Titre provenant de l'écran-titre. Bibliogr. 11 p.

Reproduction de : Thèse de doctorat : Sciences cognitives. Neurosciences cognitives : Lyon 2 : 2007. / Titre provenant de l'écran-titre. Bibliogr.

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The electrical synapse was first described over 50 years ago. Since that time appreciation of its complexity and importance has grown, including the hypothesis that early transient formation of these synapses is important to adult patterns of connectivity in neural networks. Presented in this dissertation are studies utilizing identified neurons in cell culture from the snail Helisoma trivolvis to examine discrete periods of electrical synapse formation during regeneration with sustained or transient expression. Extensive knowledge of connectivity patterns of the buccal neurons of Helisoma in cell culture and the ganglia, provide a useful framework for looking at modulation and manipulation of electrical synapses and their impact and emerging connectivity in a simple neuronal network. Two types of electrical connections were observed those that were transient, between a B19 and a B110 and those that were sustained, between a B19 and another B19. Dopamine (DA) modulation of forming electrical synapses (FES) produces a synapse specific effect at those either destined to be transient (TES) or sustained (SES) and may be a direct effect on the gap junctions at the synapses, as is the case at TES, or an indirect effect on other membrane currents, as seen in SES. DA modulation produces different outcomes at SES-centered networks and TES-centered networks with respect to new chemical synapse formation, demonstrating network-dependent effects of electrical synapse modulation. Pharmacological blockade of chemical and electrical components at forming mixed synapses in some cases alters subsequent synapse formation although due to the variable nature does not appear to be a direct interaction between chemical and electrical synapses. Three-cell networks appear to display a balancing mechanism for overall electrical coupling when electrical synapses are blocked suggesting a competition for some resource in the construction or trafficking of gap junctions. In addition to electrophysiological examinations, network coupling can be assessed utilizing fluorescent calcium imaging to look at coincidence of calcium changes as an output for coupling between cells. This technique provides a useful tool for less invasive studies of neuronal networks and the impact of coupling at mixed synapses.

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Current concepts of hippocampal circuitry assume a large population of excitatory principal neurones whose activity is largely governed by a network of local-circuit GABAergic interneurones. The diversity of hippocampal local-circuit neurones and their synaptic control over principal cell activity was investigated in vitro, in order to define their synaptic connections and functional roles. Single and dual intracellular recordings were made from local-circuit neurones and pyramidal cells in area CA1 of the rat hippocampal slice. Interneurones were tentatively distinguished from pyramidal cells based on their firing as well as their membrane properties. Intracellular labelling of recorded cells with the marker biocytin revealed a diversity of cell types based on differential dendritic and axonal morphology and synaptic connections. The physiological data revealed that all types of interneurone tested evoked inhibitory postsynaptic potentials (IPSPs) in simultaneously recorded pyramidal cells. The IPSPs had fast rise and decay kinetics and the ones tested pharmacologically, were mediated by GABA_A receptors. Similarly, individual interneurones were also shown to innervate other local-circuit interneurones in addition to pyramidal cells, the evoked effects being qualitatively similar in both types of postsynaptic targets. The postsynaptic effect and functional role of one type of hippocampal interneurone, the basket cell, was investigated in greater detail. Basket cell-evoked IPSPs were reliable, but showed some frequency-dependent attenuation. Moreover, basket cell IPSPs were found to interact with intrinsic pyramidal cell conductances to elicit rebound depolarisations and facilitate action potential generation. More detailed investigation showed that basket and axo-axonic cells were particularly effective in entraining pyramidal cell firing and sub-threshold membrane potential oscillations. Through these powerfully tuned mechanisms, sub-types of local-circuit interneurone provide a powerful mechanism to synchronise the activity of pyramidal cells. These results demonstrate a remarkable diversity of GABAergic local-circuit neurones in the hippocampal CA1 area and suggest that specific subtypes of cell mediate different functions.