Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

The world is a complex and dynamic place. The incredibly dense and constantly changing information stream with which our senses are bombarded must be decomposed, taken in, and processed by any organism hoping to make enough sense of this world in order to survive to the next moment. For complex behaviors, and in particular a great many of those that we often feel define us as a human species, this dense sensory stream must not just be processed, but the important features of the environment must be further distilled and structured into representations that can then be stored long-term to guide future behavior through the joint processes of Learning and Memory. The primary goal of this thesis is to further our understanding of the neurobiological bases - at the subcellular, circuit, and network level - of learning and memory. The hippocampus, one of the most studied systems in the brain by far, is thought to play a central role in learning and memory. Principal cells in the hippocampus become tuned to environmental features, forming persistent representations of an animal’s environment, but the precise mechanisms by which these representations are formed, used, and maintained remain unresolved. By employing a variety of experimental techniques including in vivo two-photon calcium imaging, extracellular electrophysiology, optogenetics, and chemogenetics in awake, behaving mice, we attempted to characterize the subcellular and circuit determinants of place field representations and to connect them to these cells’ role in spatial learning and memory.

No description available.

<p>The primary visual cortex (V1) is a center in the visual pathway that receives the converging information and sends diverging information to multiple visual areas. It is essential for the normal functioning of the visual system. While processing the input from the outside world, it is also continually modified by the sensory experience. This thesis is dedicated to studying the plasticity in the visual cortex that is associated with experience and brain damage recovery. In this thesis, we discovered that the visual experience induces 5 Hz oscillations that recruit inhibition in V1, sharpening the feature selectivity. We have also demonstrated that gene therapy to convert astrocytes into neurons induces neuronal circuit plasticity and functional recovery in mouse V1 following ischemia.</p>

Although inhibitory cortical interneurons play a critical role in regulating brain excitability and function, the effects of stroke on these neurons is poorly understood. In particular, interneurons expressing vasoactive intestinal peptide (VIP) specialize in inhibiting other classes of inhibitory neurons, and thus serve to modulate cortical sensory processing. To understand how stroke affects this circuit, we imaged VIP neuron responses (using GCaMP6s) to low and high intensity forepaw stimulation, both before and after focal stroke in somatosensory cortex. Our data show that the fraction of forelimb responsive VIP interneurons and their response fidelity (defined as a cell’s number of responsive trials out of eight trials at a certain imaging week) was significantly reduced in the first week after stroke, especially when lower intensity forepaw stimulation was employed. The loss of responsiveness was most evident in highly active VIP neurons (defined by their level of responsiveness before stroke), whereas less active neurons were minimally affected. Of note, a small fraction of VIP neurons that were minimally active before stroke, became responsive afterwards suggesting that stroke may unmask sensory responses in some neurons. Although VIP responses to forepaw stimulation generally improved from 2-5 weeks recovery, the variance in response fidelity after stroke was comparatively high and therefore less predictable than that observed before stroke. Lastly, stroke related changes in response properties were restricted to within 400μm of the infarct border. These findings reveal the dynamic and resilient nature of VIP neurons and suggest that a sub-population of these cells are more apt to lose sensory responsiveness during the initial phase of stroke, whereas some minimally responsive cells are progressively recruited into the forelimb sensory circuit. Furthermore, stroke appears to disrupt the predictability of sensory-evoked responses in these cortical interneurons which could have important consequences for sensory perception. / Graduate / 2021-01-13

Le cerveau est composé de réseaux de neurones interconnectés dont la mise en place au cours du développement cérébral est hautement régulée par des processus cellulaires, moléculaires et fonctionnels. Un dysfonctionnement de ces processus peut perturber l’établissement de ces réseaux et conduire à des pathologies neurologiques. L’épilepsie absence est une pathologie génétiquement déterminée qui apparait au cours de l’enfance. Les crises d’absences sont caractérisées par une altération de la conscience et par la présence de décharges de pointe-ondes (DPO) sur l’électroencéphalogramme initiées au sein d’un zone restreinte du cortex. Cependant, on sait peu de choses sur les mécanismes conduisant à la mise en place des décharges épileptiques récurrentes au cours de l’enfance (i.e. l’épileptogenèse). Nous avons fait l’hypothèse que des anomalies du processus de maturation cérébrale sont à l’origine de l’apparition des DPO.J’ai vérifié cette hypothèse chez un modèle génétique d’épilepsie absence, le rat GAERS. Dans un premier temps, j’ai étudié l’épileptogenèse du GAERS grâce à l’enregistrement in vivo du potentiel de champs local et de l’activité intracellulaire des neurones pyramidaux au niveau du site d’initiation des DPO, le cortex somatosensoriel (SoCX). Nous avons mis en évidence que les DPO se développent progressivement après la fin d’une période de maturation hautement sensible et malléable du SoCx (i.e. période critique). La maturation des décharges épileptiques consiste en une augmentation de leur fréquence, de leur durée et en l’évolution du motif de décharge jusqu’à l’âge adulte, période à laquelle ces paramètres atteignent une relative stabilité. De plus, ces changements sont associés à une altération graduelle des propriétés intrinsèques des neurones pyramidaux qui s’accompagne d’une augmentation progressive de la force de l’activité synaptique locale et d’une propension accrue des neurones du SoCx à générer des oscillations synchrones.Nous avons ensuite recherché les raisons de cette prédisposition anormale des neurones du SoCx à se synchroniser chez le GAERS. Dans ce but, nous avons cherché à mettre en évidence des anomalies de la maturation corticale au niveau de la structure et de l’organisation fonctionnelle du SoCx avant l’apparition des DPO. En combinant l’IRM, des marquages immunohistochimiques et le traçage rétrograde monosynaptique des connexions longue distance par le virus de la rage modifié, nous avons pu montrer qu’aucune anomalie globale du cerveau et du SoCx n’est présente chez le GAERS avant l’apparition des DPO. Afin de déterminer la présence d’anomalies fonctionnelles nous avons utilisé l’imagerie calcique biphoton et enregistré in vivo la dynamique de l’activité spontanée du réseau de neurones des couches 2-3 du SoCx. Chez le GAERS, nous avons mis en évidence que ces neurones sont plus actifs et dévoilent une organisation fonctionnelle différente de celle des animaux contrôles. Enfin, pour comprendre comment cette organisation fonctionnelle anormale est médiée, nous avons étudié la structure dendritique et synaptique du SoCx en combinant la microscopie électronique et la reconstruction morphologique de neurones. Nous avons mis en évidence un élargissement des épines dendritiques associé à un allongement de la densité post-synaptique au sein du SoCx chez le GAERS.L’ensemble de ces résultats démontrent la nature progressive du développement de l’épilepsie absence ainsi que l’existence d’anomalies de la maturation corticale qui affectent la structure et la fonction du réseau neuronal, avant l’apparition des crises épileptiques. Ces altérations constituent une prédisposition à l’établissement et l’évolution des DPO et sont une cible thérapeutique potentielle qui pourrait permettre de bloquer la mise en place des crises d’absences. / The brain is organized into several interconnected neuronal networks whose formation is highly regulated by cellular, molecular and functional processes. The dysfunction of these processes during brain development could disrupt neuronal circuit establishment and lead to neurological pathologies. Absence epilepsy is a genetically determined disease with a childhood onset. Absence seizures are characterized by an impairment of the consciousness associated on the electroencephalogram with spike and wave discharges (SWD). However, little is known about the mechanisms leading to the establishment of recurrent epileptic discharges (i.e. epileptogenesis). We hypothesized that SWD onset originates from an abnormal brain maturation.During my PhD, I examined this hypothesis in a recognized genetic model of absence epilepsy, the GAERS rat. First, I studied the epileptogenic process by recording in vivo the local field potential and the intracellular activities of pyramidal neurons in the initiating area of SWD, the somatosensory cortex (SoCx), at different post natal days in GAERS. We showed that SWD progressively developed after the end of a highly sensitive and plastic maturation period of the SoCx (i.e critical period). Afterward, epileptic discharges maturation consists in an increase of their duration, their number and in an evolution of the pattern reaching a relative stability at adulthood. Moreover, these changes are associated with a gradual abnormal alteration of the intrinsic properties of pyramidal neurons which is accompanied with a progressive increase in the strength of the local synaptic activity and a growing propensity of SoCx neurons to generate synchronized oscillations.Then, we explored the reasons for this abnormal susceptibility of SoCx neurons to be more synchronized in GAERS. We sought to bring to light anomalies of SoCx maturation at the structural and functional organization level prior to SWD onset in GAERS. Combining MRI, immunohistochemistry labeling and rabies-mediated retrograde monosynaptic tracing to reveal long-range connectivity, we showed that, prior to SWD onset, no brain and SoCx architecture abnormalities could be observed in GAERS. Then, using two photon calcium imaging we recorded in vivo the spontaneous activity of SoCx layers 2-3 neurons to evidence their functional organization. We found that these neurons were more active and unveiled a different functional organization in GAERS compared to control animals. Finally, to understand how is mediated this abnormal functional organization, we studied the dendritic and synaptic fine structure of SoCx neurons by combining electron microscopy and morphological neuron reconstruction. We highlighted an enlargement of the dendritic spines as well as an increase of the post-synaptic density length in the GAERS SoCx.Taken together, these findings showed the progressive nature of absence epilepsy development and the presence of abnormalities in the cortical maturation which affect the structure and the functional of the neuronal network the prior to SWD. These alterations constitute a breeding ground for the establishment and evolution of SWD. Future studies will aim at interfering with the epileptogenesis process should target these early alterations to stop seizure development.

Schizophrenia is a neurological disorder with a complex psychopathology, which is far from fully elucidated. In the patients with this disorder, changes on anatomical, cellular, and neurotransmitter level have been found. The aim of this work is to elucidate the function of specific ionotropic glutamate receptors in NG2 glia in the hippocampus of a mouse model of schizophrenia. For this purpose, a mouse model of schizophrenia was generated and validated using immunohistochemistry and behavioural testing. Mice with NG2 glia labelled by a fluorescent protein with a calcium indicator also in NG2 glia were used to observe the activity of glutamate channels and the properties of the extracellular space in these mice. Changes were found in the schizophrenic animals when compared to control animals in the numbers of hippocampal oligodendrocyte lineage cells, in prepulse inhibition and in both volume fraction and tortuosity of the extracellular space in hippocampus. Moreover, the percentage of cells responding to glutamate receptor agonists in NG2 glia in hippocampus also differed significantly between the schizophrenic and the control animals. In conclusion, it can be said that we were able to observe significant changes in the mouse model of schizophrenia that we generated in comparison to control...

The hippocampal CA1 is crucial for myriad types of learning and memory. It is theorized to provide a spatiotemporal framework for the encoding of relevant information during learning, allowing an individual to create a cognitive map of its environment and experiences. To probe CA1 network dynamics that underlie such complex cognitive function, in this work we used recently developed cellular optical imaging techniques that provide high spatial and temporal resolutions. Genetically-encoded calcium indicators offer the ability to record intracellular calcium dynamics, a proxy of neural activity, from hundreds of cells in behaving animals with single cell resolution in genetically-defined cell types. In complement, recently developed genetically-encoded voltage indicators have enabled direct recording of transmembrane voltage of individual genetically-defined cells in behaving animals. The work presented here uses the genetically-encoded calcium indicator GCaMP6f and the genetically-encoded voltage indicator SomArchon to interrogate the activities of individual hippocampal CA1 neurons and their relationship to the dynamics of the broader network during behavior. First, we provide the first in vivo, real-time evidence that two unique populations of CA1 cells encode trace conditioning and extinction learning, two distinct phases of hippocampal-dependent learning. The population of cells responsible for the representation of extinction learning emerges within one session of extinction training. Second, we perform calcium imaging in a mouse model containing a total knockout of NEXMIF, a gene causative of autism spectrum disorder. We reveal that loss of NEXMIF causes over-synchronization of the CA1 circuit, particularly during locomotion, impairing the information encoding capacity of the network. Finally, we conduct voltage imaging of CA1 pyramidal cells and parvalbumin (PV)-positive interneurons, with simultaneous recording of local field potential (LFP), to characterize how cellular-level membrane dynamics and spiking relate to network-level LFP. We demonstrate that in PV neurons, membrane potential oscillations in the theta frequency range show consistent synchrony with LFP theta oscillations and organize spike timing of the PV population relative to LFP theta, indicating that PV interneurons orchestrate theta rhythmicity in the CA1 network. In summary, this dissertation utilizes genetically-encoded optical reporters of neural activity, providing critical insights into the function of the CA1 as a flexible, diverse network of individual neurons.

Models of sensory processing have historically abstracted underlying biological circuits, due to unknown connectivity and/or complexity. In contrast, the use of tractable and anatomically well-characterized model organisms such as the fruit fly Drosophila melanogaster allows us to utilize biological constraints in models of sensory processing to understand underlying circuit mechanisms and make more accurate predictions. This approach has been used to dissect motion vision circuits, but investigations into color vision - a salient visual feature for many animals - have been limited. Here, we investigate the circuit mechanisms of the early color circuit of the fruit fly and assess its information processing capabilities. Using in vivo two-photon calcium imaging and genetic manipulations, we measure the chromatic tuning properties of photoreceptor axons and their primary targets in the medulla neuropil. At the level of photoreceptor axons, we show that opponent processes are the result of a dual mechanism - a direct pathway specific to insect physiology and an indirect pathway found across the animal kingdom. Both pathways are necessary to decorrelate incoming signals and efficiently represent chromatic information. We built an anatomically constrained model that is able to quantitatively reproduce these color opponent responses without fitting synaptic weights. Instead, we used electron-microscopy-derived synaptic count, an anatomically defined measure, as a proxy for synaptic weight, thereby linking structure to function. Downstream of photoreceptors, we find that neurons shift their tuning and become highly selective for particular directions in color space - similar to “hue-selective” neurons in primate cortex. To achieve this selectivity, these neurons require input from all types of photoreceptors and an interneuron that determines the neuron's preferred chromatic direction. We extended our anatomically constrained model to incorporate these downstream neurons and are able to predict their responses, qualitatively and quantitatively.In summary, the detailed reconstruction of the fly circuit anatomy predicts the mechanisms of multiple stages of color information processing and allows us to infer functional roles for each part of the circuit. The circuit motifs, we uncover, are shared across species and hint at convergent mechanisms that underlie the transformation from an opponent neural code to a hue selective code.

No description available.