The amygdala is crucially involved in the acquisition and retention of fear memories. Experimental research on fear conditioning has shown that memory retrieval shortly followed by pharmacological manipulations or extinction, thereby interfering with memory reconsolidation, decreases later fear expression. Fear memory reconsolidation depends on synaptic plasticity in the amygdala, which has been demonstrated in rodents using both pharmacological manipulations and retrieval-extinction procedures. The retrieval-extinction procedure decreases fear expression also in humans, but the underlying neural mechanism have not been studied. Interfering with reconsolidation is held to alter the original fear memory representation, resulting in long-term reductions in fear responses, and might therefore be used in the treatment of anxiety disorders, but few studies have directly investigated this question. The aim of this thesis was to examine the effects of the retrieval-extinction procedure on amygdala activity and behavioral fear expression in humans. The work presented here also investigated whether findings from studies on recent fear memories, established through fear conditioning, extends to naturally occurring long-term phobic fears. Study I, combining fear conditioning and a retrieval-extinction procedure with functional magnetic resonance imaging (fMRI), demonstrated that memory retrieval shortly followed by extinction reduces later amygdala activity and fear expression in healthy subjects. In Study II, these subjects were re-tested 18 months later. The results showed that the effects on fear expression were still present and that initial amygdala activity predicted long-term fear expression. Using an adapted version of the retrieval-extinction procedure, Study III showed that memory retrieval shortly followed by exposure to spider pictures, attenuates subsequent amygdala activity and increases approach behavior in subjects with life-long fear of spiders. In Study IV, these subjects were re-tested 6 months later, and the results showed that effects on amygdala activity as well as approach behavior were maintained. In summation, retrieval-extinction leads to long-lasting reductions in amygdala activity and fear expression. These findings are consistent with the hypothesis that retrieval-extinction alters an amygdala dependent fear memory. Retrieval-extinction can also attenuate long-term phobic fears, indicating that this manipulation could be used to enhance exposure-based treatments for anxiety disorders.

Fear memories, here defined as learned associations between a stimulus and a physiological fear reaction, are formed through fear conditioning. In animals, fear memories, present in the lateral amygdala, undergo reconsolidation after recall. Moreover, this reconsolidation process can be disrupted both pharmacologically and behaviourally, resulting in a reduced fear response to the stimulus. This thesis examines the attenuation of fear memories by disrupting reconsolidation in humans, using measures of both the central and peripheral nervous system activity. Serotonergic and dopaminergic genes have previously been tied to both fear conditioning and anxiety disorders, where fear conditioning mechanisms are important. In order to evaluate the possible role of fear memory reconsolidation mechanims in the effect on fear and anxiety by these genes, this thesis also compare the reconsolidation disruption effect between different serotonergic and dopaminergic genotypes. Study I examined the attentuation of fear memories by disrupting reconsolidation in humans using reacquisition as a measure of the return of fear. Moreover, study I investigated the impact of differences in serotonergic and dopaminergic alleles on this process. Study II examined the attentuation of fear memories by disrupting reconsolidation in humans using reinstatement as a measure of the return of fear. Study II also investigated the impact of differences in serotonergic and dopaminergic alleles on the process of fear memory reconsolidation. Study III used psychophysiology and fMRI to localize the functional neural activity mediating the fear memory reconsolidation disruption effect. In summary, this thesis provides evidence that fear memories are attenuated by reconsolidation disruption in humans and that serotonergic and dopaminergic alleles influence this process. Moreover, this thesis support that human fear memory reconsolidation is amygdala-dependent, suggesting an evolutionary shared memory mechanism.

La neurogenèse hippocampique adulte fait référence à la création de neurones durant la vie adulte dans le gyrus denté de l’hippocampe. Une décennie de recherche a démontré l’importance de cette neurogenèse chez l’adulte dans les processus de mémoire. En particulier, la neurogenèse adulte est nécessaire à l’apprentissage spatial et l’apprentissage spatial lui-même augmente la survie et accélère le développement d’une population de nouveaux neurones immatures. Cependant, l’implication de ces nouveaux neurones « sélectionnés » par l’apprentissage dans le devenir de la mémoire reste incertaine. En conséquence, le travail de cette thèse porte sur l’étude du rôle de ces nouveaux neurones dans les processus de mémoire spatiale à long terme résultants de l’apprentissage d’origine, comme la restitution et la reconsolidation de la mémoire. En effet depuis plus d’un siècle, on sait qu’un apprentissage n’induit pas immédiatement une mémoire stable. Les souvenirs sont tout d’abord fragiles, puis vont au fil du temps devenir stables et insensibles aux perturbations via un processus appelé «consolidation de la mémoire». Cependant ce processus n’est pas immuable ; les souvenirs établis peuvent à nouveau devenir labiles lorsqu'ils sont rappelés ou réactivés lors d’une restitution de la mémoire. Cette déstabilisation d’une mémoire consolidée nécessite alors un nouveau processus de stabilisation appelé « reconsolidation de la mémoire ». Depuis sa découverte, la reconsolidation a vivement intéressé le milieu de la recherche sur la mémoire et un nombre croissant d’études a cherché à comprendre les mécanismes sous-tendant cette reconsolidation, en particulier dans l'hippocampe. Étonnamment, le processus de reconsolidation n’a été que très peu envisagé dans le contexte de la neurogenèse hippocampique adulte.Nous avons tout d’abord mis au point un protocole de reconsolidation de la mémoire spatiale du rat dans le labyrinthe aquatique de Morris. Cela nous a permis de montrer que les néo-neurones nés avant l’apprentissage étaient activés lors de la reconsolidation de la mémoire spatiale, ce qui n’est pas le cas des neurones issus du développement précoce. Afin de pouvoir établir une relation de causalité entre néo-neurones et processus de reconsolidation, nous avons ensuite développé un outil basé sur la technique pharmacogénétique des DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) couplés à un rétrovirus. Cet outil permet de marquer les néo-neurones à leur naissance et de les manipuler (inhiber ou stimuler l’activation) plus tard, lors des processus de mémoire à long terme. Nous avons observé que les néo-neurones immatures modifiés par l’apprentissage étaient non seulement activés par la reconsolidation mais également nécessaire à celle-ci, à l’inverse des néo-neurones matures au moment de l’apprentissage. Nous avons enfin montré que stimuler l’activité des néo-neurones au moment de la restitution de la mémoire améliorait les performances des rats dans le labyrinthe aquatique.Ensemble, ces résultats de thèse soulignent le rôle critique de la neurogenèse hippocampique adulte dans la stabilisation de la mémoire spatiale à long terme. / Adult hippocampal neurogenesis refers to the creation of neurons during adult life in the dentate gyrus of the hippocampus. A decade of research has demonstrated the importance of this adult neurogenesis in memory processes. In particular, adult neurogenesis is necessary for spatial learning and spatial learning itself increases survival and accelerates the development of a population of new immature neurons. However, the involvement of these new modified / promoted / amplified / selected neurons by learning in the fate of memory remains unclear. The work of this thesis focuses on the study of the role of these new neurons in the long-term spatial memory processes resulting from the original learning, such as retrieval and reconsolidation.For more than a century, we know that learning does not immediately induce a stable memory. Memories are fragile at first and then become stable and insensitive to interferences over time, through a process called “memory consolidation". However this process is not immutable; the established memories can become labile again when they are reactivated during memory recall. This destabilization of a consolidated memory requires then a new stabilization process called "memory reconsolidation". Since its discovery, the reconsolidation process has strongly interested the memory research community and a growing number of studies have sought to understand the mechanisms underlying this reconsolidation, particularly in the hippocampus. Surprisingly, the process of reconsolidation has rarely been considered in the context of adult hippocampal neurogenesis.We first developed a protocol for memory reconsolidation of spatial memory in the Morris water maze in rats. This allowed us to show that new neurons born before learning were activated during reconsolidation of spatial memory, which is not the case of the neurons generated during the early development. In order to establish a causal relationship between new neurons and reconsolidation, we developed a tool based on the pharmacogenetic technique of DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) coupled with a retrovirus. This tool is used to tag new neurons at their birth and manipulate them (inhibit or stimulate their activation) later during long-term memory processes. We observed that the population of neurons that were immature at the time of learning are not only activated by but also necessary for reconsolidation, unlike new neurons that were mature at the time of learning. We have finally shown that stimulating the activity of new neurons during retrieval improves the performance of rats in the water maze.All together, these thesis results highlight the critical role of adult hippocampal neurogenesis in long-term spatial memory stabilization.

Rationale and objective: Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are disorders of learning and memory that often occur comorbidly. Exposure to trauma-related cues can increase alcohol intake in PTSD patients that are using alcohol to self-medicate. The recurrence of anxiety symptoms with subsequent alcohol use may initiate a destructive cycle where stress and alcohol exposure impair the function of the prefrontal cortex (PFC). While the incidence of these disorders has steadily increased, current therapies and treatments often lack efficacy. Thus, investigation into the underlying neurocircuitry responsible for the establishment and maintenance of these disorders is necessary to develop novel treatment targets. Methods: The present study examined the effects of ethanol exposure on the ability to create new learned associations around previously conditioned fear cues in a rat model. Animals were exposed to fear conditioning followed by chronic intermittent ethanol to translationally model trauma exposure followed by alcohol abuse. Optogenetics was used to inhibit the prelimbic (PrL) or infralimbic (IfL) cortex during fear memory reconsolidation, and fear behaviors were measured during subsequent extinction and spontaneous recovery tests. Results and conclusion Chronic ethanol exposure led to deficits in fear extinction learning and increased freezing during spontaneous recovery, both of which were prevented following inhibition of the PrL, but not the IfL, during memory reconsolidation. These results support the involvement of the PrL in fear learning and memory, and strongly suggest that the PrL could serve as a potential target for the treatment of the learning and memory deficits that occur following exposure to stress and alcohol.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The reactivation of a memory results in its destabilization, requiring a process of memory reconsolidation to maintain it. Spermidine is an endogenous aliphatic amine with polycationic structure that modulates N-methyl-D-aspartate (NMDA) receptor activity and improves memory. Recent evidence suggests that systemic administration of spermidine improves the reconsolidation of fear memory. Here we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by intrahippocampal (ih) administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4 mA footshock as unconditioned stimulus. Twenty-four hours after training, animals were re-exposed to the apparatus in the absence of shock (reactivation session). Immediately after the reactivation session, spermidine (2-200 ρmol/site); the PKC inhibitor, 3-[1- (dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl) maleimide hydrochloride (GF 109203X, 0.3 - 30 ρg/site); the antagonist of the polyamine-binding site at the NMDA receptor, arcaine (0.2 - 200 ρmol/site) or the PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.02 - 2 nmol/site) were injected intra-hipocampally (i.h.). Testing was carried out in the same apparatus, twenty-four hours after reactivation. Freezing scores at testing were considered a measure of memory. While the post-reactivation administration of spermidine (20 and 200 ρmol/site) improved, GF 109203X (1, 10 and 30 ρg/site) impaired memory reconsolidation. GF 109203X (0.3 ρg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. The post-reactivation administration of arcaine (200 pmol/site) impaired and PMA (2 nmol/site) improved memory reconsolidation. PMA (0.2 nmol/site) prevented arcaine (200 ρmol/site)-induced impairment of memory reconsolidation. The protein synthesis inhibitor anisomycin (2 μg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. These drugs had no effect on memory if they were administered in the absence of reactivation. These results suggest that the enhancement of memory reconsolidation induced by the administration of spermidine involves PKC activation. / A reativação de uma memória resulta na sua desestabilização, que exigem um processo de reconsolidação de memória para mantê-la. A espermidina é uma amina alifática endógena com estrutura policatiônica que modula a atividade do receptor Nmetil- D-aspartato (NMDA) e melhora a memória. Evidências recentes sugerem que a administração sistêmica de espermidina melhora a reconsolidação da memória de medo. No entanto não se sabe se a administração intra hipocampal de espermidina melhora a reconsolidação da memória e nem se a proteína cinase dependente de cálcio (PKC) e síntese proteica estão envolvidas neste efeito. Portanto, no presente estudo verificou-se o envolvimento da PKC e da síntese proteica na melhora da reconsolidação da memória do medo induzida pela administração intrahipocampal (ih) de espermidina em ratos. Ratos Wistar machos foram treinados na tarefa de medo condicionado contextual utilizando-se choque de 0,4 mA como estímulo incondicionado. Vinte e quatro horas após o treino, os animais foram re-expostos ao aparelho na ausência de choque (sessão reativação). Imediatamente após a sessão de reativação foram administradas, por via ih, espermidina (2-200 ρmol/sítio); o inibidor da PKC, 3- [1- (dimetilaminopropil) indol-3-il] -4- (indol-3-il) maleimida (GF 109203X, 0,3-30 ρg/sítio); o antagonista do sítio de ligação das poliaminas no receptor de NMDA, arcaína (0,2-200 ρmol/sítio) ou o ativador de PKC, 12-miristato 13-acetato de forbol (PMA, 0,02-2 nmol/sítio). Os testes foram realizados no mesmo aparelho, 24 horas após a sessão de reativação. O tempo de imobilidade dos animais durante o teste foi considerado como medida de memória. Enquanto a administração de espermidina (20 e 200 ρmol/sítio) melhorou, GF 109203X (1, 10 e 30 ρg/sítio) prejudicou a reconsolidação da memória. O GF 109203X (0,3 ρg/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). A administração da arcaína (200 pmol/sítio) prejudicou e PMA (2 nmol/sítio) melhorou a reconsolidação da memória. O PMA (0,2 nmol/sítio) preveniu o prejuízo na reconsolidação da memória induzido por arcaína (200 ρmol/sítio). O inibidor de síntese de proteica, anisomicina (2 ug/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). Estas drogas não tiveram nenhum efeito sobre a memória quando foram administrados na ausência da sessão de reativação. Estes resultados sugerem que a melhora da reconsolidação da memória induzida pela administração ih de espermidina envolve a síntese proteica e a ativação de PKC.