The central nervous system (CNS) is protected by a vascular blood-brain barrier that prevents many types of pathogens from entering the brain. Still, some pathogens have evolved mechanisms to traverse this barrier and establish a long-term infection. The apicomplexan parasite, Toxoplasma gondii, is one such pathogen with the ability to infect the CNS in virtually all warm-blooded animals, including humans. Across the globe, an estimated 30% of the human population is infected with Toxoplasma, an infection for which mounting evidence suggests increases the risk for developing neurological and neuropsychiatric disorders, like seizures and schizophrenia. In my dissertation, I investigate the telencephalic neural circuit changes induced by long-term Toxoplasma infection in the mouse brain and the neuroimmune signaling role of the complement system in mediating microglial remodeling of neural circuits following parasitic infection. While there has been keen interest in investigating neural circuit changes in the amygdala – a region of the brain involved in fear response and which Toxoplasma infection alters in many species of infected hosts – the hippocampus and cortex have been less explored. These are brain regions for which Toxoplasma also has tropism, and moreover, are rich with fast-spiking parvalbumin perisomatic synapses, a type of GABAergic synapse whose dysfunction has been implicated in epilepsy and schizophrenia. By employing a range of visualization techniques to assess cell-to-cell connectivity and neuron-glia interactions (including immunohistochemistry, ultrastructural microscopy, and microglia-specific reporter mouse lines), I discovered that longterm Toxoplasma infection causes microglia to target and ensheath neuronal somata in these regions and subsequently phagocytose their perisomatic inhibitory synapses. These findings provide a novel model by which Toxoplasma infection within the brain can lead to seizure susceptibility and a wider range of behavioral and cognitive changes unrelated to fear response. In the Toxoplasma infected brain, microglia, along with monocytes recruited to the brain from the periphery, coordinate a neuroinflammatory response against pathogenic invasion. This is characterized by a widespread activation of these cells and their increased interaction with neurons and their synaptic inputs. Yet, whether T. gondii infection triggers microglia and monocytes (i.e. phagocytes) to target, ensheath, and remove perisomatic inhibitory synapses on neuronal somata indiscriminately, or whether specificity exists in this type of circuit remodeling, remained unclear. Through a comprehensive assessment of phagocyte interactions with cortical neuron subtypes, I demonstrate that phagocytes selectively target and ensheath excitatory pyramidal cells in long-term infection. Moreover, coupling of in situ hybridization with transgenic reporter lines and immunolabeling revealed that in addition to phagocytes, excitatory neurons also express complement component C3 following infection (while inhibitory interneurons do not). Lastly, by employing targeted deletion of complement components, C1q and C3, I show that complement is required for phagocyte ensheathment of excitatory cells and the subsequent removal of perisomatic inhibitory synapses on these cells (albeit not through the classical pathway). Together, these studies highlight a novel role for complement in mediating synapse-type and cell-type specific circuit remodeling in the Toxoplasma infected brain. / Doctor of Philosophy / Parasites are microorganisms that rely on other living organisms (called hosts) for their survival. Although some parasites only live on their hosts, others have developed ways to establish infections and obtain the nutrients that keep them alive from host cells. My Ph.D. research has focused on studying one of these parasites, Toxoplasma gondii (commonly referred to as Toxo), that has evolved the unique ability to establish brain infections in almost all animals around the world, from rodents to humans. Recent discoveries show that brain infection with this parasite can cause seizures, an imbalance in the way that specialized cells of the brain (called neurons) communicate with each other, causing harmful hyperactivity within the brain. Toxo infection can also cause behavioral and cognitive changes in infected animals, making them more susceptible to predation. In humans, infection with Toxo increases their risk for developing different types of mental illness, such as schizophrenia. The focus of my Ph.D. research has been in trying to understand, at the cellular and molecular level, how infection with this parasite can lead to seizures and behavioral changes, by using mice as a model. Mice have a similar brain structure to humans, and over the years, scientists have developed many tools that allow us to visualize and study the connections between neurons (called synapses). I'm interested in understanding how changes in these connections affect how neurons communicate with each other, and ultimately, how we behave and think. I have been studying a type of connection that, if lost or damaged, can lead to seizures and some types of mental illness. These connections are called 'perisomatic inhibitory synapses', and they form on many distinct types of neurons, but specifically on the cell bodies of these neurons. They act as a traffic light, informing neurons when and for how long to 'slow down' their activity. I discovered that after the parasite enters the brain, it causes another type of cell in the brain, called microglia, to extensively interact with neurons in the cortex and hippocampus (areas of your brain important for thinking, executing behavior, and learning). Microglia are immune cells of the brain that inspect the brain for anything damaged or that doesn't belong (like parasites) and removes them from the brain. By performing experiments where I delete individual immune molecules from mice, I found that one immune molecule, called 'complement component C3' acts as cue for microglia to find these cells, wrap around them, and permanently remove these important connections. Surprisingly, however, microglia don't remove these connections from all neurons, indiscriminately, they do so only on one specific cell type called 'excitatory pyramidal neurons,' and as the name implies, they're the ones who drive activity in the brain. My half-a-decade's worth of research helps us understand parasitic infections in the brain in a couple of ways: First, I have discovered one of the mechanisms by which neuronal connections are lost in the Toxo-infected brain (which is a mechanism that leads to loss of neuronal connections in the injured and aging brain as well). This is significant because it might provide insight into why some people who are infected with Toxo develop seizures or mental illness, while others don't. More importantly, Toxo-infection causes changes in the brain that are very specific, in terms of both the type of neuronal connection that is affected and the type of cell that is affected. Why these changes are so specific remain to be uncovered, but it suggests that Toxo can either a) trigger a unique immune response in the brain that leads to very precise changes in neuron-toneuron connections and signaling or b) the parasite, while hiding inside of neurons, may hijack the machinery of certain cell types in a way that helps them survive longer.

Les processus d'apprentissage et de mémoire sont contrôlés par des circuits et éléments neuronaux spécifiques. De nombreuses études ont récemment mis en évidence que les circuits corticaux jouent un rôle important dans la régulation des comportements de peur, cependant, leurs caractéristiques anatomiques et fonctionnelles restent encore largement inconnues. Au cours de ma thèse, en utilisant des enregistrements unitaires et des approches optogénétiques chez la souris libre de se comporter, nous avons pu montrer que les interneurones inhibiteurs du cortex auditif et du cortex préfrontal médian forment un microcircuit désinhibiteur permettant respectivement l'acquisition et l'expression de la mémoire de peur conditionnée. Dans les deux cas, les interneurones parvalbuminergiques constituent l'élément central du circuit et sont inhibés de façon phasique. D’un point de vue fonctionnel, nous avons démontré que cette inhibition était associée à la désinhibition des neurones pyramidaux par un mécanisme de réduction de l'inhibition continue exercée par les interneurones parvalbuminergiques. Ainsi, les interneurones parvalbuminergiques peuvent contrôler temporellement l'excitabilité des neurones pyramidaux. En particulier, nous avons montré que l'acquisition de la mémoire de peur conditionnée dépend du recrutement d'un microcircuit désinhibiteur localisé dans le cortex auditif. En effet, au cours du conditionnement de peur, la présentation du choc électrique induit l'inhibition des interneurones parvalbuminergiques, ce qui a pour conséquence de désinhiber les neurones pyramidaux du cortex auditif et de permettre l’apprentissage du conditionnement de peur. Dans leur ensemble, ces données suggèrent que la désinhibition est un mécanisme important dans l'apprentissage et le traitement de l'information dans les circuits corticaux. Dans un second temps, nous avons montré que l'expression de la peur conditionnée requière l'inhibition phasique des interneurones parvalbuminergiques du cortex préfrontal médian. En effet, leur inhibition désinhibe les cellules pyramidales préfrontales et synchronise leur activité en réinitialisant les oscillations thêta locales. Ces résultats mettent en évidence deux mécanismes neuronaux complémentaires induits par les interneurones parvalbuminergiques qui coordonnent et organisent avec précision l’activité neuronale des neurones pyramidaux du cortex préfrontal pour contrôler l'expression de la peur conditionnée. Ensemble, nos données montrent que la désinhibition joue un rôle important dans les comportements de peur en permettant l’association entre des informations comportementalement pertinentes, en sélectionnant les éléments spécifiques du circuit et en orchestrant l'activité neuronale des cellules pyramidales. / Learning and memory processes are controlled by specific neuronal circuits and elements. Numerous recent reports highlighted the important role of cortical circuits in the regulation of fear behaviour, however, the anatomical and functional characteristics of their neuronal components remain largely unknown. During my thesis, we used single unit recordings and optogenetic manipulations of specific neuronal elements in behaving mice, to show that both the auditory cortex and the medial prefrontal cortex contain a disinhibitory microcircuit required respectively for the acquisition and the expression of conditioned fear memory. In both cases, parvalbumin-expressing interneurons constitute the central element of the circuit and are phasically inhibited during the presentation of the conditioned tone. From a functional point of view, we demonstrated that this inhibition induced the disinhibition of cortical pyramidal neurons by releasing the ongoing perisomatic inhibition mediated by parvalbumin-expressing interneurons onto pyramidal neurons. Thereby, this disinhibition allows the precise temporal regulation of pyramidal neurons excitability. In particular, we showed that the acquisition of associative fear memories depend on the recruitment of a disinhibitory microcircuit in the auditory cortex. Fear-conditioning-associated disinhibition in auditory cortex is driven by foot-shock-mediated inhibition of parvalbumin-expressing interneurons. Importantly, pharmacological or optogenetic blockade of pyramidal neuron disinhibition abolishes fear learning. Together, these data suggest that disinhibition is an important mechanism underlying learning and information processing in cortical circuits. Secondly, in the medial prefrontal cortex, we demonstrated that expression of fear behaviour is causally related to the phasic inhibition of prefrontal parvalbumin-expressing interneurons. Inhibition of parvalbumin-expressing interneuron activity disinhibits prefrontal pyramidal neurons and synchronizes their firing by resetting local theta oscillations, leading to fear expression. These results identify two complementary neuronal mechanisms both mediated by prefrontal parvalbumin-expressing interneurons that precisely coordinate and enhance the neuronal efficiency of prefrontal pyramidal neurons to drive fear expression. Together these data highlighted the important role played by neuronal disinhibition in fear behaviour by binding behavioural relevant information, selecting specific circuit elements and orchestrating pyramidal neurons activity.