The cerebral cortex is the largest subdivision of the human brain and is associated with higher cognitive functions. These functions are based on the interconnections between the neurons that form pre- and postnatally in the different telencephalic regions. The processes of neurons with similar functions and connectivity follow the same course and form axon tracts. There are three main axons tracts analysed in this thesis the corpus callosum, the corticothalamic/thalamocortical tracts and the lateral olfactory tract that transfers olfactory information to the telencephalon. In the mouse, these tracts are generated during embryogenesis as axons project to their target area. The mechanisms by which axons navigate still need to be elucidated. Studies of a number of mutant mice have shown that axon pathfinding is under the control of genes. Gli3 is a zinc finger transcription factor with known roles in axon pathfinding. Gli3 is widely expressed in progenitor cells of the dorsal and ventral telencephalon complicating the elucidation of the molecular mechanisms by which Gli3 controls axon tract formation. My aim here is to investigate the spatial and temporal requirements for Gli3 in axon pathfinding in the forebrain using Gli3 conditional mutants as a tool. Regarding the corpus callosum, my findings demonstrated a crucial role for Gli3 in the dorsal telencephalon, but not in the septum or medial ganglionic eminence, to control corpus callosum formation and indicated that defects in the formation of the corticoseptal boundary affect the positioning of callosal guidepost cells. Moreover, conditional inactivation of Gli3 in dorsal telencephalic progenitors led to few corticothalamic axons leaving the cortex in a restricted lateral neocortical domain. This restricted entry is at least partially caused by an expansion of the piriform cortex, which forms from an enlarged progenitor domain of the ventral pallium. Transplantation experiments showed that the expanded piriform cortex repels corticofugal axons. Moreover, expression of Sema5B, a chemorepellent for corticofugal axons produced by the piriform cortex, is similarly expanded. Hence, control of lateral cortical development by Gli3 at the progenitor level is crucial for corticothalamic pathfinding. Finally, by using Emx1Cre;Gli3fl/fl mutants I analysed the consequences of the expansion of the piriform cortex on the formation of the lateral olfactory tract (LOT). This analysis showed that LOT axons also appear to be medially shifted with LOT collaterals aberrantly colonising the expanded piriform cortex. Time course analysis confirmed an expansion of the paleocortical primordium from E13.5 onwards, coinciding with the arrival of the LOT axons. Hence, it is possible that the expanded piriform cortex contributed to the medial shift of the LOT. In conclusion, these findings support a strong link between Gli3 controlled early patterning defects and axon pathfinding defects and form the basis for future analysis of the molecular mechanisms by which Gli3 controls axon pathfinding in the forebrain. My findings also reveal how alterations in GLI3 function may contribute to connectivity defects in human patients with mutations in GLI3.

The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

Our laboratory has invented a series of oxime acetylcholinesterase (AChE) reactivators (US Patent 9,227,937) that enter the brain, reduce time to cessation of seizure-like activities, and prevent organophosphorus compound (OP) neuropathology, not seen with the current U.S. approved AChE reactivator, pralidoxime (2-PAM). Thus, 2-PAM fails to protect the brain against damage and long-term cognitive and behavioral deficits seen in humans after OP exposure. However, the mechanisms by which these novel oximes provide central neuroprotection through preservation of neuronal cell structures from damage in a rat model are not fully understood by AChE reactivation alone. This dissertation investigated neurotoxic mechanisms of NIMP as potential targets for additional direct and indirect neuroprotection by our lead in vivo AChE reactivator, Oxime 20. Male Sprague Dawley rats exposed to NIMP experienced neurotoxic effects in areas critical to OP-induced seizure generation (e.g., hippocampus and piriform cortex) such as the inhibition of multiple serine hydrolases (i.e., fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL)), necrotic cell death evident by increased necrotic receptor-interacting serine/threonine-protein kinase 1 (RIPK1) levels and no apoptotic caspase-3 activity, and increased levels of neuroinflammation via elevated levels of pro-inflammatory oxylipins 4 days post lethal exposure. However, due to the lack of statistical significance, NIMP exposure did not definitively affect the subcellular location of either phosphorylated excitatory N-methyl-D-aspartate (NMDA) receptor or inhibitory γ-aminobutyric acid (GABA) receptor subunits. Results suggested that Oxime 20 therapy provided neuroprotection after NIMP exposure, such as limited reactivation of other serine hydrolase targets, significantly decreased RIPK1 levels (i.e., necrotic environment) in the hippocampus, and significantly decreased inflammatory oxylipins 4 days post-NIMP exposure. Thus, reducing OP-induced neuroinflammation might be the main contributor to the neuroprotection (i.e., neuronal cell structure preservation) previously observed in our laboratory.

Comment les souvenirs olfactifs sont-ils stockés dans notre cerveau? Plusieurs études suggèrent que le cortex olfactif (piriforme) jouerait un rôle important dans la mémoire olfactive. J'ai donc voulu déterminer si cette région du cerveau est impliquée dans le rappel d'un souvenir olfactif. Pour cela, j'ai manipulé l'activité des neurones du cortex piriforme ayant été actifs pendant un apprentissage olfactif aversif, en utilisant cfos comme marqueur d'activation neuronale. Plus précisément, j'ai choisi de travailler avec la lignée de souris transgénique cfos-tTA (et de manière moins développée avec la lignée fosCreERT2) et d'utiliser les récepteurs "DREADDs". J'ai montré que lors du test de mémoire, l'inactivation des neurones du cortex piriforme actifs pendant l'apprentissage rend la souris amnésique. De manière réciproque, la réactivation artificielle de ces neurones récupère le souvenir de l'association odeur-stimulus aversif. Ainsi, une trace mnésique se forme au niveau du cortex piriforme, et plus précisément au niveau des neurones actifs lors de l'apprentissage. Ce résultat ouvre la voie à de nombreux champ d'investigations pour mieux comprendre les mécanismes de la mémoire. J'ai choisi d'explorer de manière préliminaire l'effet de l'apprentissage sur la représentation des odeurs au niveau du cortex piriforme. En parallèle, en utilisant une approche théorique, j'ai étudié si un modèle basé sur les variations de potentiel de membrane dendritique pouvait prédire l'occurrence d'un type de plasticité à une synapse corticale. L'ensemble de ces travaux ont permis d'accroître nos connaissances sur le fonctionnement de la mémoire olfactive. / Olfaction is an evolutionarily old sensory modality that plays key roles in the survival of many species and is densely interwoven with memory and emotions. However, how odor memories are formed and stored in the brain remains largely unknown. To address these questions, we studied the olfactory (piriform) cortex of mice, which is a good candidate for encoding olfactory memory traces. We used c-fos as a marker of neural activity and the cfos-tTA transgenic mouse line (and the fosCreERT2 mouse line to a lesser extent) to selectively express chemogenetic receptors (DREADDs) in piriform neurons that are active during an olfactory fear conditioning task. We found that chemogenetically reactivating these ensembles artificially retrieves the memory while chemogenetically silencing them impairs memory retrieval. Piriform neurons active during olfactory learning thus play a key role in memory retrieval. These results open new horizons in understanding memory trace formation. We decided to explore in a preliminary way how learning shapes piriform network properties. In parallel, using a theoretical framework, we investigated if a model based on dendritic voltage could predict synaptic plasticity. Taken together, these experiments will provide important insights into the mechanisms of odor coding and memory.

Dans le cerveau, les fonctions de la phosphatase alcaline non spécifique des tissus (TNAP) ne sont pas clairement identifiées. La localisation et l'expression de cette enzyme au niveau neuronal suggère cependant, qu'elle joue un rôle important dans le développement et le fonctionnement du cerveau. Cela est supporté par la présence de graves crises d'épilepsie chez les humains porteurs d'une mutation de la TNAP. Ces crises d'épilepsie sont létales chez les souris KO pour la TNAP. Des études chez la souris montrent que la TNAP pourrait réguler l'inhibition postsynaptique médiée par le GABA et elle pourrait être impliquée dans l'inhibition présynaptique médiée par l'adénosine. L'adénosine est, en partie, synthétisée via la déphosphorylation successive de l'ATP en ADP puis en AMP par des ectonucléotidases. Parmi elles, la TNAP et l'ecto- 5'-nucléotidase (NT5E) catalysent l'hydrolyse de l'AMP en adénosine dans le cortex cérébral. L'adénosine agit principalement au niveau présynaptique par l'intermédiaire des récepteurs A1. Ainsi l'adénosine a une influence sur la transmission synaptique et sur la plasticité synaptique. Ceci pourrait expliquer, en partie, les crises d'épilepsie observées chez les souris KO pour la TNAP. Les deux objectifs principaux de ma thèse ont été : (1) évaluer la contribution de la TNAP dans la production d'adénosine dans le cerveau ; (2) étudier l'influence de l'adénosine sur la plasticité synaptique. Premièrement, l'étude de la contribution de la TNAP dans la production d'adénosine dans le cerveau a été réalisée au moyen de deux approches complémentaires. Une approche métabolomique (spectroscopie RMN du proton) sur des cerveaux entiers de souris KO pour la TNAP a permis de montrer que la TNAP participe, entre autre, à la synthèse d'adénosine dans le cerveau. Une deuxième approche, électrophysiologique sur tranches de cerveaux de souris in vitro, nous permet d'examiner les conséquences de l'inhibition des ectonucléotidases intervenant dans la synthèse de l'adénosine. Elle a révélé que l'inhibition des ectonucléotidases (TNAP et NT5E) ne supprime pas l'effet inhibiteur de l'AMP médiée par les récepteurs A1. Deuxièmement, nous avons étudié l'influence de l'adénosine sur la plasticité synaptique à courte terme. Nous avons enregistré des potentiels de champs dans la couche Ia du cortex piriforme en réponse à des stimulations électriques (3,125 à 100 Hz) présentée avec des fréquences recouvrant la gamme d'oscillations physiologiques. Nos résultats montrent qu'avec de fortes concentrations d'adénosine, la facilitation est accentuée par rapport à celle observée en situation contrôle. Cet effet est observé pour des fréquences supérieures ou égales à 25 Hz. De plus, cette accentuation est d'autant plus grande que la fréquence est élevée (maximum atteint à 100 Hz pour 100 µM). En bloquant l'action de l'adénosine endogène, l'effet contraire est observé : une facilitation déficitaire par rapport au contrôle et dont le défaut est croissant avec la fréquence de stimulation. Tous ces résultats convergent vers l'hypothèse qu'une déficience en TNAP, traduite par une absence d'adénosine, pourrait contribuer au maintien des processus épileptiques générés par un déséquilibre de l'inhibition et de l'excitation dû à une diminution de GABA. L'effet inhibiteur de l'AMP médié par les récepteurs A1 ne serait pas suffisant pour contrecarrer les crises d'épilepsie observées chez les sujets hypophosphatasiques et les souris KO pour la TNAP. / The functions of Tissue Nonspecific Alkaline Phosphatase (TNAP) in the brain are not clearly identified. The localization and expression of TNAP at the neuronal level, however, suggests that it plays a prominent role in the development and the function in the brain. This is supported by the presence of severe epileptic seizures in humans carrying TNAP mutation. These epileptic seizures are lethal in TNAP KO mice. Studies in mice show that TNAP could regulate GABA-mediated postsynaptic inhibition and may be involved in presynaptic inhibition mediated by adenosine. Adenosine is, partly, synthesized via the successive dephosphorylation of ATP to ADP and then to AMP by ectonucleotidases. Among them TNAP and ecto-5'-nucleotidase (NT5E) are able to hydrolyze AMP into adenosine. Adenosine acts mainly at the presynaptic level via A1 receptors activation. Adenosine has an influence on synaptic transmission and thus on synaptic plasticity. This could partly explain the epileptic seizures observed in TNAP knock-out mice. The two main purposes of my thesis were: (1) to evaluate the contribution of TNAP in adenosine production in the brain; (2) to study the influence of adenosine on synaptic plasticity. Firstly, the study of the contribution of TNAP in adenosine production in the brain was carried out using two complementary approaches. A metabolomic approach (proton NMR spectroscopy) on whole brains of TNAP KO mice showed that TNAP in involved in adenosine synthesis in the brain. In a second approach, in vitro electrophysiological recordings on mouse brain slices allowed us to examine the consequences of the inhibition of the ectonucleotidases involved in adenosine synthesis. This revealed that inhibition of ectonucleotidases (TNAP and NT5E) did not suppress the inhibitory effect of AMP mediated by A1 receptors. Secondly, we studied the influence of adenosine on short-term synaptic plasticity. Field potentials were recorded in response to electrical stimulations (3.125 to 100 Hz) applied with frequencies encompassing the range of physiological oscillation. Our results show that, with high adenosine concentrations, the facilitation is emphasized compared to that observed in the control situation. This effect is observed for frequencies greater than or equal to 25 Hz. In addition, the higher the frequency, the greater the facilitation. Finally, by blocking the action of endogenous adenosine, the opposite effect was observed: a deficient facilitation with respect to the control, whose defect was increasing with stimulation frequency. All these results converge towards the hypothesis that TNAP deficiency, expressed by absence of adenosine, could contribute to the maintenance of the epileptic processes generated by an imbalance of the neuronal inhibition and the excitation due to a decrease of GABA. AMP inhibitory effect mediated by A1 receptors, would not be sufficient to counteract epileptic seizures observed in hypophosphatasic patients and TNAP KO mice.

Les comportements olfactifs nécessitent de reconnaitre les odeurs sur une large gamme de concentration tout en restant sensible aux changements de concentration. Pour accomplir cette tâche paradoxale, le système olfactif doit façonner des représentations des odeurs qui soient à la fois dépendantes et indépendantes de leurs concentrations.Nous avons combiné des techniques de génétique murine, microscopie biphotonique, et enregistrements neurophysiologiques extracellulaires pour caractériser l’activité neuronale en réponse aux odeurs dans le bulbe olfactif et le cortex olfactif (piriforme) de la souris. En utilisant une souris au « nez monoclonal », nous montrons que les circuits du bulbe olfactif sont capables d’amplifier les entrées sensorielles très faibles, et d’atténuer les entrées envahissantes. En revanche, nous observons que le niveau d’activité neuronale induite par les odeurs dans le cortex piriforme est globalement indépendant de leurs concentrations. En outre, nous avons identifié une sous-population de neurones du cortex piriforme encodant l’identité d’une odeur indépendamment de sa concentration. Nos résultats d’imagerie calcique in vivo et d’enregistrement neurophysiologiques suggèrent que cette invariance à la concentration dans le cortex piriforme est assurée par les interneurones positifs à la paravalbumine, une sous-population de neurones inhibiteurs.Ces résultats mettent en évidence deux étapes différenciées du traitement de l’information dans les voies neurales de l’olfaction : tandis que le bulbe olfactif normalise les entrées sensorielles, les microcircuits du cortex piriforme intègrent cette information en composantes sensorielles distinctes. / Olfactory behaviors require the identification of odors across a large range of different concentrations, yet are exquisitely sensitive to changes in odor concentrations. To accomplish this seemingly paradoxical task the olfactory system must generate odor representations that are, at once, both concentration-dependent and concentration-invariant.We have used a combination of mouse genetics, in vivo two-photon microscopy, and extracellular multielectrode recording techniques to characterize odor-evoked activity in the olfactory bulb and olfactory (piriform) cortex of mice. Taking advantage of a mouse with a “monoclonal nose” in which the sensory input map is strongly perturbed, we show that olfactory bulb circuits are able to amplify very weak sensory inputs, and to suppress pervasive input.In contrast, we found that the overall level of odor-evoked neural activity in the piriform cortex is largely concentration-invariant. Moreover, we identified a small subpopulation of odor-responsive piriform neurons, which encodes odor identity independent of concentration. In vivo calcium imaging and extracellular multielectrode recordings suggest that parvalbumin-expressing interneurons, a subpopulation of inhibitory neurons, mediate concentration invariance in the piriform cortex.These results highlight contrasting processing mechanisms of sensory information along the olfactory pathway: while the olfactory bulb normalizes sensory inputs, microcircuits of the piriform cortex integrate this information into distinct sensory features. This process may allow for the simultaneous representation of identity and intensity in the olfactory system.

SCHIP-1 est une protéine cytoplasmique enrichie aux nœuds de Ranvier et aux segments initiaux des axones matures, où elle est associée à l’ankyrine G. SCHIP-1 est également exprimée dans le système nerveux central pendant le développement embryonnaire. Nous montrons ici que les souris mutées pour Schip1 présentent des anomalies morphologiques de la commissure antérieure formée par les axones du cortex piriforme, du noyau olfactif antérieur et de l’amygdale. Ces anomalies résultent de défauts de croissance et de guidage axonal in vivo au cours du développement. Les neurones du cortex piriforme d’embryons mutés présentent un retard d’initiation et de croissance axonales, et des anomalies de guidage axonal in vitro. Des expériences de vidéomicroscopie montrent que SCHIP-1 régule la réponse des cônes de croissance à la molécule de guidage EphB2, importante pour le développement de la commissure antérieure. Les souris mutées présentent en outre une diminution de l’épaisseur du cortex piriforme qui affecte spécifiquement les couches de neurones de projection. Cette diminution résulterait d’une augmentation de la mort cellulaire et non d’un défaut de génération ou de migration des neurones. De manière intéressante, ces anomalies morphologiques sont associées à des comportements anormaux qui pourraient reposer sur des défauts d’intégration des odeurs. Le cortex piriforme joue un rôle-clé dans la discrimination, l’association et l’apprentissage des odeurs. Les souris mutées pour Schip1 semblent donc être un modèle prometteur pour étudier la fonction du cortex piriforme ainsi que celle de la commissure antérieure, peu connues à ce jour. / SCHIP-1 is a cytoplasmic component of nodes of Ranvier and axon initial segments of mature axons, where it associates with ankyrinG. SCHIP-1 is also expressed in the CNS during mouse early embryonic stages. Here we report that Schip1 mutant mice display morphological abnormalities of the anterior commissure, which is composed of axons from piriform cortex, anterior olfactory nucleus, and amygdala. These abnormalities are due to impaired axon elongation and navigation in vivo during development. Piriform cortex neurons display axon initiation/outgrowth delay and guidance defects in vitro. Time-lapse imaging indicates that SCHIP-1 regulates the response of growth cones to EphB2, a guidance cue important for anterior commissure development. Besides, mutant mice display a reduced thickness of the piriform cortex, which affects projection neuron layers, and is likely to result from cell death rather than from impairment of pyramidal neuron generation or migration. Interestingly these morphological defects are associated with abnormal behavior related to defects in odor processing. The piriform cortex is thought to play a key role in odor discrimination, association and learning. Thus Schip1 mutant mice appear to be an interesting model to further characterize piriform cortex as well as anterior commissure functions, which are yet poorly known.

Um dos modelos experimentais mais utilizados para estudar a epilepsia do lobo temporal (ELT) é o abrasamento (kindling) por estimulação elétrica diária da amígdala, o abrasamento elétrico convencional. Uma alternativa rápida e eficaz a esse modelo é o abrasamento elétrico rápido, também capaz de gerar crises límbicas, porém com 10 estímulos elétricos aplicados ao dia, por 2 dias. No 3º dia é aplicado um estímulo elétrico adicional, o 21º estímulo, quando podem ser testadas drogas antiepilépticas ou estudados mecanismos de plasticidade ou memória. Entre as principais áreas ativadas nas crises límbicas encontram-se o complexo amigdalóide, a formação hipocampal, o córtex piriforme e neocórtices adjacentes. O envolvimento de estruturas olfatórias na ELT é antigo e estudos indicam que a exposição a um estímulo olfatório é capaz de suprimir, inibir ou induzir a ocorrência de crises. Todas as evidências clínicas e experimentais dão suporte científico para a hipótese de que a estimulação olfatória com o 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), uma potente substância química, derivada das fezes de raposa e que biologicamente representa o cheiro de predador pode influenciar no processo de crises evocadas por estimulação elétrica da amígdala. O objetivo geral do presente estudo foi avaliar a influência da apresentação do estímulo olfatório com TMT nas crises epilépticas de ratos Wistar, submetidos ao abrasamento elétrico rápido da amígdala. Para tanto, os parâmetros químicos do TMT foram avaliados, bem como as respostas comportamentais de ratos Wistar machos naives submetidos ao estímulo olfatório com diferentes doses de TMT. Na sequência, um novo grupo de ratos Wistar machos naives foi submetido ao protocolo de abrasamento elétrico rápido da amígdala com a aquisição dos registros eletrencefalográficos (EEGráficos) do córtex piriforme, formação hipocampal além do complexo amigdalóide. Após abrasados os animais foram expostos ao TMT ou água destilada, previamente ao 21º estímulo elétrico. Posteriormente o tecido cerebral foi processado (perfundido, crioprotegido, congelado e cortado) e então foram feitas as técnicas histoquímicas de: Nissl e Fluoro-Jade C (FJC, marcador de neurodegeneração). As respostas comportamentais foram analisadas mediante o uso do Índice de Gravidade para Crises Límbicas e da neuroetologia. Adicionalmente foi avaliada a expressão EEGráfica do 1º, 20º e 21º estímulos e verificada a presença/ausência de neurodegeneração em regiões do sistema límbico. Os resultados da análise comportamental obtidos nesse estudo foram comparados com os obtidos no protocolo de estimulação olfatória com TMT nas crises audiogênicas agudas de ratos da cepa WAR. O TMT desencadeou reações de medo e modificou as sequências comportamentais, reduziu a atividade motora e os comportamentos de autolimpeza. Dados qualitativos da cromatografia gasosa e algoritmos matemáticos possibilitaram estabelecer as concentrações na câmara para as diferentes doses de TMT. Além disso, a cromatografia gasosa identificou que 30 minutos é o tempo necessário para saturação e dessaturação da câmara ao TMT, e indicou uma saturação homogênea do interior dessa câmara. O TMT puro no abrasamento elétrico rápido em ratos Wistar foi capaz de reduzir significativamente o Índice de Gravidade para Crises Límbicas comparado à água, corroborando os dados neuroetológicos que indicam o efeito supressor do TMT nas crises, tanto para o modelo de abrasamento elétrico rápido quanto para as crises audiogênicas agudas. Os resultados da duração da pós-descarga EEGráfica primária no 21º estímulo foram inconclusivos, sendo necessárias outras análises empregando diferentes métodos analíticos. Com a técnica de FJC não foi possível verificar morte celular por necrose em qualquer região cerebral avaliada. / One of the most widely used experimental models to study temporal lobe epilepsy (TLE) is the kindling by electrical daily stimulation of the amygdala, the conventional kindling. A rapid and effective alternative to this model is rapid electrical kindling, also capable of generating limbic seizures, but with 10 electrical stimuli applied per day for 2 days. On the 3rd day an additional electrical stimulus is applied, the 21st stimulus, when antiepileptic drugs can be tested or mechanisms of plasticity and memory can be studied. Among the main areas activated in limbic seizures are the amygdaloid complex, the hippocampal formation, piriform cortex and adjacent neocortices. The involvement of the olfactory structures in TLE is old and studies indicate that exposure to an olfactory stimulus is capable to suppress or inhibit or induce the occurrence of seizures. All the clinical and experimental evidences provide scientific support for the hypothesis that the olfactory stimulation with 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), a powerful chemical substance derived from fox feces which biologically represents the \"predator smell can influence the seizures process evoked by electrical stimulation of the amygdala. The overall objective of this study was to evaluate the influence of olfactory stimulation with TMT in seizures of Wistar rats subjected to rapid electrical kindling of the amygdala. Therefore, the chemical parameters of TMT were evaluated, as well as behavioral responses of naive male Wistar rats exposed to the olfactory stimulus with different concentrations of TMT. Other group of rats was electrically stimulated in the amygdaloid complex, following the protocol of rapid electrical kindling and the electroencephalographic recordings (EEGraphic) obtained from the piriform cortex, hippocampal formation in addition to the amygdaloid complex. After scorched the animals were exposed to TMT or distilled water, prior to the 21st electrical stimulation. Subsequently the cerebral tissue was processed (perfused, cryoprotected, frozen and sliced) and then processed for Nissl and Fluoro-Jade C histochemistry (FJC, a marker of neurodegeneration). The behavioral responses were analyzed by using the Severity Index for Limbic Seizures and neuroethology. In addition to EEG, reviewed after the 1st, 20th and 21th stimuli we also examined the presence/absence of neurodegeneration in regions of the limbic system. The results obtained in this study were compared with those obtained in the protocol of olfactory stimulation with TMT on acute audiogenic seizures of rats from the WAR strain. The TMT triggered fear reactions and modified the behavioral sequences, reduced motor activity and grooming behavior. Qualitative data from gas chromatography and mathematical algorithms made possible to establish the concentrations in the camera for the different doses of TMT. In addition, the gas chromatography helped to identify that 30 minutes is the time required for saturation and desaturation of the camera to TMT and indicated a homogeneous saturation of the interior of such camera. The pure TMT in rapid electrical kindling in Wistar rats was able to significantly reduce the Severity Index for Limbic Seizures, compared to water, corroborating the data of the neuroethology method indicating the suppressive effect of TMT in seizures, in both, the model of rapid electrical kindling as well as the acute audiogenic seizures. However, the results of the duration of the EEGraphic primary after-discharge at the 21th stimulus were inconclusive, requiring further analysis using different analytical methods. With the technique of FJC it was not observed necrotic cell death in any studied brain region.

Um dos modelos experimentais mais utilizados para estudar a epilepsia do lobo temporal (ELT) é o abrasamento (kindling) por estimulação elétrica diária da amígdala, o abrasamento elétrico convencional. Uma alternativa rápida e eficaz a esse modelo é o abrasamento elétrico rápido, também capaz de gerar crises límbicas, porém com 10 estímulos elétricos aplicados ao dia, por 2 dias. No 3º dia é aplicado um estímulo elétrico adicional, o 21º estímulo, quando podem ser testadas drogas antiepilépticas ou estudados mecanismos de plasticidade ou memória. Entre as principais áreas ativadas nas crises límbicas encontram-se o complexo amigdalóide, a formação hipocampal, o córtex piriforme e neocórtices adjacentes. O envolvimento de estruturas olfatórias na ELT é antigo e estudos indicam que a exposição a um estímulo olfatório é capaz de suprimir, inibir ou induzir a ocorrência de crises. Todas as evidências clínicas e experimentais dão suporte científico para a hipótese de que a estimulação olfatória com o 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), uma potente substância química, derivada das fezes de raposa e que biologicamente representa o cheiro de predador pode influenciar no processo de crises evocadas por estimulação elétrica da amígdala. O objetivo geral do presente estudo foi avaliar a influência da apresentação do estímulo olfatório com TMT nas crises epilépticas de ratos Wistar, submetidos ao abrasamento elétrico rápido da amígdala. Para tanto, os parâmetros químicos do TMT foram avaliados, bem como as respostas comportamentais de ratos Wistar machos naives submetidos ao estímulo olfatório com diferentes doses de TMT. Na sequência, um novo grupo de ratos Wistar machos naives foi submetido ao protocolo de abrasamento elétrico rápido da amígdala com a aquisição dos registros eletrencefalográficos (EEGráficos) do córtex piriforme, formação hipocampal além do complexo amigdalóide. Após abrasados os animais foram expostos ao TMT ou água destilada, previamente ao 21º estímulo elétrico. Posteriormente o tecido cerebral foi processado (perfundido, crioprotegido, congelado e cortado) e então foram feitas as técnicas histoquímicas de: Nissl e Fluoro-Jade C (FJC, marcador de neurodegeneração). As respostas comportamentais foram analisadas mediante o uso do Índice de Gravidade para Crises Límbicas e da neuroetologia. Adicionalmente foi avaliada a expressão EEGráfica do 1º, 20º e 21º estímulos e verificada a presença/ausência de neurodegeneração em regiões do sistema límbico. Os resultados da análise comportamental obtidos nesse estudo foram comparados com os obtidos no protocolo de estimulação olfatória com TMT nas crises audiogênicas agudas de ratos da cepa WAR. O TMT desencadeou reações de medo e modificou as sequências comportamentais, reduziu a atividade motora e os comportamentos de autolimpeza. Dados qualitativos da cromatografia gasosa e algoritmos matemáticos possibilitaram estabelecer as concentrações na câmara para as diferentes doses de TMT. Além disso, a cromatografia gasosa identificou que 30 minutos é o tempo necessário para saturação e dessaturação da câmara ao TMT, e indicou uma saturação homogênea do interior dessa câmara. O TMT puro no abrasamento elétrico rápido em ratos Wistar foi capaz de reduzir significativamente o Índice de Gravidade para Crises Límbicas comparado à água, corroborando os dados neuroetológicos que indicam o efeito supressor do TMT nas crises, tanto para o modelo de abrasamento elétrico rápido quanto para as crises audiogênicas agudas. Os resultados da duração da pós-descarga EEGráfica primária no 21º estímulo foram inconclusivos, sendo necessárias outras análises empregando diferentes métodos analíticos. Com a técnica de FJC não foi possível verificar morte celular por necrose em qualquer região cerebral avaliada. / One of the most widely used experimental models to study temporal lobe epilepsy (TLE) is the kindling by electrical daily stimulation of the amygdala, the conventional kindling. A rapid and effective alternative to this model is rapid electrical kindling, also capable of generating limbic seizures, but with 10 electrical stimuli applied per day for 2 days. On the 3rd day an additional electrical stimulus is applied, the 21st stimulus, when antiepileptic drugs can be tested or mechanisms of plasticity and memory can be studied. Among the main areas activated in limbic seizures are the amygdaloid complex, the hippocampal formation, piriform cortex and adjacent neocortices. The involvement of the olfactory structures in TLE is old and studies indicate that exposure to an olfactory stimulus is capable to suppress or inhibit or induce the occurrence of seizures. All the clinical and experimental evidences provide scientific support for the hypothesis that the olfactory stimulation with 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), a powerful chemical substance derived from fox feces which biologically represents the \"predator smell can influence the seizures process evoked by electrical stimulation of the amygdala. The overall objective of this study was to evaluate the influence of olfactory stimulation with TMT in seizures of Wistar rats subjected to rapid electrical kindling of the amygdala. Therefore, the chemical parameters of TMT were evaluated, as well as behavioral responses of naive male Wistar rats exposed to the olfactory stimulus with different concentrations of TMT. Other group of rats was electrically stimulated in the amygdaloid complex, following the protocol of rapid electrical kindling and the electroencephalographic recordings (EEGraphic) obtained from the piriform cortex, hippocampal formation in addition to the amygdaloid complex. After scorched the animals were exposed to TMT or distilled water, prior to the 21st electrical stimulation. Subsequently the cerebral tissue was processed (perfused, cryoprotected, frozen and sliced) and then processed for Nissl and Fluoro-Jade C histochemistry (FJC, a marker of neurodegeneration). The behavioral responses were analyzed by using the Severity Index for Limbic Seizures and neuroethology. In addition to EEG, reviewed after the 1st, 20th and 21th stimuli we also examined the presence/absence of neurodegeneration in regions of the limbic system. The results obtained in this study were compared with those obtained in the protocol of olfactory stimulation with TMT on acute audiogenic seizures of rats from the WAR strain. The TMT triggered fear reactions and modified the behavioral sequences, reduced motor activity and grooming behavior. Qualitative data from gas chromatography and mathematical algorithms made possible to establish the concentrations in the camera for the different doses of TMT. In addition, the gas chromatography helped to identify that 30 minutes is the time required for saturation and desaturation of the camera to TMT and indicated a homogeneous saturation of the interior of such camera. The pure TMT in rapid electrical kindling in Wistar rats was able to significantly reduce the Severity Index for Limbic Seizures, compared to water, corroborating the data of the neuroethology method indicating the suppressive effect of TMT in seizures, in both, the model of rapid electrical kindling as well as the acute audiogenic seizures. However, the results of the duration of the EEGraphic primary after-discharge at the 21th stimulus were inconclusive, requiring further analysis using different analytical methods. With the technique of FJC it was not observed necrotic cell death in any studied brain region.