Дисертації з теми "Animals, Astrocyte"

Thesis (Ph. D.)--Ohio State University, 2004.
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Les astrocytes représentent une des populations cellulaires les plus nombreuses du cerveau. Ces cellules gliales extrêmement ramifiées y jouent un rôle essentiel, notamment dans le cortex cérébral, où elles forment un réseau tridimensionnel continu tout en présentant une hétérogénéité importante au niveau morphologique, moléculaire et fonctionnel. Afin de déterminer comment le réseau astrocytaire est établi au cours du développement cortical murin, des analyses clonales ont été effectuées grâce à une stratégie de marquage multicolore permettant d’étudier simultanément la descendance de nombreux progéniteurs. Les résultats de ces travaux montrent que les clones d’astrocytes corticaux s’imbriquent avec leurs voisins et présentent une variabilité importante au niveau de leur composition en termes de nombre et sous-types cellulaires, leur organisation et leur dispersion spatiale. Le réseau astrocytaire se développe au cours d’une première phase dynamique de prolifération et de dispersion pendant la première semaine postnatale, suivie par une phase de maturation à l’échelle de la cellule avec augmentation du volume des astrocytes et de la complexité de leur arborisation. Ces travaux montrent par ailleurs la contribution non négligeable de progéniteurs postnataux au réseau astrocytaire, qui s’ajoute à celle des cellules souches neurales corticales embryonnaires. La grande variabilité du réseau astrocytaire à l’échelle clonale suggère que son développement repose sur des unités clonales plastiques composées de cellules dont l’organisation spatiale et les caractéristiques finales dépendent probablement de leurs interactions avec leur environnement neuronal via des acteurs moléculaires qui restent à caractériser
Astrocytes are one of the most numerous cell types in the brain. They consist in ramified glial cells that play essential roles in neural tissue where they form an uninterrupted tridimensional network, while displaying important local heterogeneity in terms of morphology and molecular marker expression. To determine how this network is established during development, multiclonal lineage tracing was performed to analyzed large numbers of astrocyte clones issued from nearby mouse cortical progenitors. Results show that cortical astrocyte clones intermix with their neighbors, display extensive variability in terms of spatial organization, numbers and subtypes of generated cells, and increase in size towards the upper part of the cortex. Furthermore, this organization develops through two stages that comprise a dynamic phase of proliferation accompanied by spatial dispersion, and a maturation phase where morphological complexity and volume increase at the single cell level. Moreover a significant contribution of subependymal postnatal progenitors to the generation of astrocytes, independent of their subtype and location, was uncovered in addition to prenatal delaminating apical progenitors. Thus cortical astrocyte network development appears unstereotyped at the clonal level. This suggests that the construction of this network relies on plastic clonal units issued from non-specified astrocyte progenitors that differentially expand and mature, and whose descendants probably acquire their final characteristics through interactions with their neuronal environment through molecular mechanisms that still need to be defined

Orientador: Alexandre Leite Rodrigues de Oliveira
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Durante o curso da encefalomielite autoimmune experimental ocorre uma grave redução das funções motoras e sensitivas. Esses eventos têm sido classicamente atribuídos ao processo desmielinizante da doença. Em ratos, os sinais clínicos da doença desaparecem 5 dias após completa tetraplegia, indicando que o processo desmielinizante não é a única causa da rápida evolução da doença. Assim sendo, investigamos as alterações sinaptológicas e o processo inflamatório induzidos pela encefalomielite autoimune experimental (EAE) em motoneurônios medulares e sua relação com o surto e remissão da doença. Para esse estudo, foram utilizados ratos Lewis, fêmeas de 7 semanas. Os animais foram induzidos à EAE por meio de dose única de proteína básica de mielina emulsificada com adjuvante completo de Freund e sacrificados no 13º dia após indução (surto grau 3) e no 26º dia (remissão da doença). Também, para investigar a possibilidade de que o tratamento com acetato de glatirâmer, uma droga imunomoduladora baseada na estrutura de aminoácidos da proteína básica de mielina, interfira no processo de plasticidade sináptica, os animais foram induzidos à EAE, tratados com AG diariamente e sacrificados após 2 semanas. Os grupos experimentais foram divididos em: estudo da aposição sináptica durante surto e remissão da doença e tratamento dos animais induzidos à EAE com AG. Assim, os espécimes foram processados para análise através de imunohistoquímica e microscopia eletrônica de transmissão. Nossos resultados indicaram que os componentes gliais (astrócitos e microglia), estimulados pela inflamação, desempenham papel ativo no processo de retração sináptica em motoneurônios alfa. Apresentamos evidências de que a eliminação de terminais sinápticos contribui para a perda da função motora observada no curso da doença e que o imunomodulador AG não só possui efeito antiinflamatório, mas também influencia diretamente na plasticidade de elementos neurais no microambiente medular. Reforçam, também, que um processo agudo de inflamação pode colaborar diretamente para a recuperação e sobrevivência neuronal, uma vez que as células inflamatórias produzem citocinas e fatores neurotróficos no microambiente medular
Abstract: During the course of experimental autoimmune encephalomyelitis, a massive loss of motor and sensitive function occurs, which has been classically attributed to the demyelination process. In rats, the clinical signs disappear within 5 days following complete tetraplegia, indicating that demyelination might not be the only cause for the rapid evolution of the disease. The immunomodulador glatiramer acetate (GA) has been shown significantly reduce the seriousness of the symptoms during the exacerbation of the disease. However, little is known about its effects on the spinal motoneurons and on their afferents. The present work investigated the occurrence of experimental autoimmune encephalomyelitis-induced changes of the synaptic covering of spinal motoneurons during exacerbation and after remission and investigated whether GA has a direct influence on synapse plasticity and on the deafferentiation of motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after fifteen days of treatment. For the both cases the spinal cords was processed for immunohistochemical analysis (IH) and electron transmission microscopy. The terminals were typed with transmission electron microscopy as C-, F- and Stype. Immunohistochemical analysis of synaptophysin, glial fibrillary acidic protein and the microglia/macrophage marker F4/80 were also used in order to draw a correlation between the synaptic changes and the glial reaction. The ultrastructural analysis showed that, during exacerbation, there was a strong retraction of both F- and S-type terminals. In this sense, both the covering as well as the length of the remaining terminals suffered great reductions. However, the retracted terminals rapidly returned to apposition, although the mean length remained shorter. A certain level of sprouting may have occurred as, after remission, the number of F-terminals was greater than in the control group. The immunohistochemical analysis showed that the peak of synaptic loss was coincident with an increased macro- and microglial reaction. Interestingly, although the GA treatment preserved synaptophysin labelling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Our results suggest that the major changes occurring in the spinal cord network during the time course of the disease may contribute significantly to the origin of the clinical signs as well as help to explain their rapid recovery and that the immunomodulator GA has a direct influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis
Doutorado
Anatomia
Doutor em Biologia Celular e Estrutural

La Leucoencefalopatia Megalencefàlica amb quists subcorticals, també anomenada MLC, és un tipus rar de leucodistròfia vacuolitzant. Actualment encara es desconeix el mecanisme fisiopatològic de la malaltia, i per tant ni hi ha cap tractament possible per als pacients. S’han descrit dos gens implicats en la malaltia MLC. El primer gen descobert s’anomena MLC1 i codifica per una proteïna de membrana que porta el mateix nom. El segon gen s’anomena GLIALCAM i codifica per una proteïna transmembrana de tipus I que també porta el mateix nom. S’ha decrit que la proteïna GlialCAM actua com a subunitat ß de MLC1 ja que es capaç de dirigir-la i concentrar-la a les unions cel•lulars. Per altra banda, GlialCAM també s’ha descrit com a subunitat auxiliar del canal de Cl- ClC-2 ja que és capaç de modificar les propietats d’activació i rectificació del canal. En la present tesi s’han generat i estudiat diferents models animals i cel•lulars per a l’estudi de la malaltia. En primer lloc, s’ha generat i s’ha caracteritzat un model de ratolí knock-out per a Mlc1. Gràcies a aquest model s’ha observat que la proteïna MLC1 és únicament astrocitària i que la proteïna GlialCAM no es independent de MLC1, ja que en absència d’aquesta es troba deslocalitzada en el cerebel. També s’ha pogut descriure per primer cop la implicació del canal de Cl- ClC-2 en la fisiopatologia, ja que els seus nivells de proteïna disminuixen en el cerebel i el canal es troba gairebé inactiu en els oligodendròcits de l’animal knock-out. Les característiques fenotípiques que presenta el model de ratolí equivalen a les característiques observades en els pacients en fases inicials de la malaltia, ja que l’animal tot i que mostra presència de vacuoles no presenta deteriorament motor i macrocefàlia aparent. També s’ha generat un model de peix zebra knock-out per a zmlc1. Aquest model presenta avantatges respecte el ratolí, com per exemple el baix cost o l’aplicació de tècniques genètiques a gran escala. Aquest model ha permés observar de nou que realment GlialCAM necessita a MLC1 per a la seva correcta localització. També s’ha observat que l’ortòleg zGlialCAMa conserva la seva funció de entre espécies ja que també es capaç de modificar les corrents de ClC-2. Aquests resultats obtinguts amb els models s’han pogut comparar amb el cervell d’una pacient. Aquest cervell demostra que MLC1 és necessària per a la correcta localització de GlialCAM en la regió del cerebel. Per altra banda, s’han desenvolupat diferents models cel•lulars. Primerament s’han estudiat els astròcits del ratolí knock-out. Aquestes cel•lules mancades de MLC1 també presenten vacuoles per tot el citoplasma, però no mostren canvis en la localització ni en els nivells de proteïna de GlialCAM i ClC-2. Aquest fet juntament amb altres estudis del grup van fer pensar si la condició necessària per a que es veguessin afectades aquestes proteïnes estaria relacionada amb el procés del sifoneig de K+. Estudis realitzats en astròcits de rata demostren que en condicions d’un alt contingut de K+, com per exemple durant una alta activitat neuronal, GlialCAM i ClC-2 és localitzen juntament a les membranes cel•lular i ClC-2 canvia les seves propietat de canal. Paral•lelament, estudis realitzats en oligodendròcits de rata també demostren que aquest fet també succeix en aquest tipus cel•lular.
Megalencefalic leukoencephalopathy with subcortical cysts, also known as MLC, is a rare type of leukodystrophy. Currently still unknown pathophysiological mechanism of the disease, and therefore there is no effective treatment possible for patients. There are two genes involved in the MLC disease. Gene was first discovered was MLC1 and this encodes for a membrane protein with the same name. The second gene is called GLIALCAM and encodes for a transmembrane protein type I that also carries the same name. In our group is has been described that GlialCAM acts as a protein ß subunit of MLC1 because it is able to direct and concentrate in the cellular junctions. Moreover, GlialCAM also act as auxiliary subunit of CLC-2 Cl channel as it is capable of modifying the activation and rectification properties of the channel. In this work we have developed two different models to study the physiopathology. The results show that GlialCAM affected by the absence of MLC1. It has been also demonstrated that ClC-2 is implicated in the disease.These results were compared with a patient brian and has been shown that MLC1 is important for the correct location of GlialCAM in the cerbellum. Have also been developed a different cellular models. The results with this models show that GlialCAM and ClC-2 could have a functional role in the process of potassium siphoning.

Dans le cadre de l'étude de l'auto-immunité dirigée contre un antigène du système nerveux, nous avons analysé, au moyen de lignées de souris transgéniques: i) les conséquences cliniques et histologiques de réactions auto-immunes induites par des lymphocytes T (LT) CD8+ spécifiques d'un antigène exprimé par des cellules gliales, les astrocytes et les cellules gliales entériques (CGE) ; ii) le rôle des LT CD8+ dans la surveillance immune du système nerveux central et l'induction de réponses inflammatoires au sein de cet organe ; et iii) les mécanismes d'induction de tolérance affectant des populations de LT CD4+ spécifiques d'un auto-antigène exprimé par les astrocytes et les CGE
We have studied autoimmunity targeting a nervous system-specific antigen with the use of several transgenic mouse lines, and have investigated : i) clinical and histopathological effects of autoimmune processes induced by CD8+ T-cells specific for a glial antigen expressed in astrocytes and enteric glial cells (EGC) ; ii) the role of CD8+ T-cells in the immune surveillance of the central nervous system and the induction of inflammatory lesions in this organ ; and iii) the mechanisms of tolerance affecting populations of astrocytes- and EGC-specific CD4+ T-cells

L'objectif de ce travail est d'explorer en détail le métabolisme de lignées astrocytaires dérivant de la moelle épinière d'embryons de rat. La respiration mitochondriale, certains paramètres de la glycolyse et l'activité de l'oncogène Akt sont étudiés dans les lignées en passages précoces (EP) et dans les lignées en passages tardifs (LP), qui se sont spontanément transformées après avoir été maintenues pendant plus de 35 passages dans le milieu de culture ne contenant pas de pyruvate. Dans les LP, en comparaison avec les EP, il existe une diminution de la glycolyse, une réduction du nombre de mitochondries par cellule et un déficit de la respiration portant sur les complexes I et II+III de la chaîne respiratoire mitochondriale. Le traitement des LP par le pyruvate, pendant 20 passages supplémentaires, ne modifie pas l'état de transformation des cellules. Alors que ce traitement rétablit la glycolyse, aucun effet bénéfique n'est constaté sur le déficit respiratoire. Le traitement des EP par du pyruvate, jusqu'à ce qu'elles soient par définition en passages tardifs, prévient la transformation spontanée. Alors que les EP traitées par le pyruvate ont un déficit modéré de la respiration, elles renferment 2 fois plus de mitochondries par cellule que les EP non traitées. L'augmentation du nombre de mitochondries compense le déficit modéré de la respiration afin de maintenir les capacités d'oxydation dans ces lignées tardives qui ne sont pas transformées. Dans l'ensemble des lignées, l'activité de l'oncogène Akt varie dans le même sens que la glycolyse et que la respiration et est directement stimulée par le pyruvate dans les lignées traitées. En conclusion, ce travail ouvre de nouvelles perspectives quant à la compréhension des mécanismes possiblement associés à la transformation astrocytaire et met en évidence de nouvelles voies thérapeutiques potentielles afin d'améliorer le contrôle des proliférations astrocytaires.

Les mécanismes physiopathologiques responsables des complications neurologiques de l'alcoolisme sont très complexes. Le métabolisme oxydatif de l'éthanol a largement été étudié au niveau du foie, mais beaucoup moins au niveau cérébral. Le but de notre travail a été d'étudier la capacité du cerveau à produire d'une part de l'acétaldéhyde et d'autre part des radicaux libres par métabolisation de l'éthanol en travaillant sur un modèle de culture de cellules astrocytaires de rat. Après avoir mis au point et valide un système d'exposition à l'éthanol des cultures de cellules en permettant d'éviter son évaporation, nous avons pu démontrer que les astrocytes sont capables de métaboliser l'éthanol en acétaldéhyde via la catalase et constituent également un site de production d'un radical libre dérivé de l'éthanol : le radical -hydroxyéthyle. Compte tenu du fait que les astrocytes jouent un rôle primordial dans le développement et le fonctionnement du système nerveux central, la preuve qu'ils sont le siège d'une production d'acétaldéhyde et de radicaux libres présentant tous les deux des effets cytotoxiques, constitue un élément important dans la compréhension des mécanismes de la neurotoxicité de l'éthanol.

A doença de Parkinson (DP) apresenta declínio da capacidade respiratória. Estudo utilizando um modelo de DP induzido pela injeção de 6-hidroxidopamina (6-OHDA) no estriado mostrou redução no número de neurônios envolvidos no controle da respiração. O objetivo desse estudo foi avaliar alterações gliais no núcleo retrotrapezóide (RTN), núcleo do trato solitário, complexo pré-Bötzinger e grupamento respiratório ventrolateral rostral (rVRG) utilizando o mesmo modelo experimental. Em ratos Wistar que receberam a injeção de 6-OHDA bilateralmente no estriado observou-se redução na imunorreatividade para proteína ácida fibrilar glial em astrócitos no rVRG a partir de 30 dias, no RTN a partir de 40 dias e nas demais regiões a partir de 60 dias. Ainda, observamos o aumento da proteína ligante de actina cruzada em microglias na região do RTN aos 30 e 40 dias. Nossos dados sugerem que a redução astrocitária contribui para as alterações respiratórias observadas neste modelo experimental e a presença de neuroinflamação no RTN pode contribuir para a perda celular nesta região.
Parkinson\'s disease (PD) has a decline in respiratory capacity. A study using a model of PD induced by the injection of 6-hydroxydopamine (6-OHDA) in the striatum showed a reduction in the number of neurons involved in the control of breathing. The aim of this study was to evaluate glial changes in the retrotrapezoid nucleus (RTN), nucleus of the solitary tract, pre-Bötzinger complex and rostral ventral respiratory group (rVRG) using the same experimental model. Wistar rats that received bilateral 6-OHDA injection in the striatum present a reduction in immunoreactivity for glial fibrillary acidic protein in astrocytes in the rVRG from 30 days, in the RTN from 40 days and in the other regions from 60 days. We also observed an increase of the Ionized calcium binding adaptor molecule 1 in microglia in the region of the RTN at 30 and 40 days. Our data suggest that astrocytic reduction contributes to the respiratory changes observed in this experimental model and the presence of neuroinflammation in the RTN may contribute to the cellular loss in this region.

Le développement des cellules gliales (astrocytes et oligodendrocytes) et l'influence de deux facteurs de la matrice extra-cellulaire: l'hyaluronane, glycosaminoglycane non sulfaté de haut poids moléculaire, et l'hyaluronectine, protéine se liant spécifiquement à l'hyaluronane, ont été étudiés dans des cultures de cerveaux de rats nouveau-nés. Nous avons montré que l'hyaluronane est sécrété par les astrocytes de type 1; qu'il est un marqueur des cellules immatures de la lignée oligodendrogliale et des astrocytes de type 2, mais n'est pas trouvé sur les oligodendrocytes matures; qu'il est un inhibiteur spécifique de la prolifération des cellules immatures et de la formation des oligodendrocytes s'opposant en particulier aux effets du facteur de croissance dérivé des plaquettes, à la différence des autres glycosaminoglycanes qui n'ont pas cet effet. L'hyaluronectine n'est pas trouvée sur les cellules gliales immatures mais est un marqueur des oligodendrocytes matures et de leurs précurseurs immédiats par lesquels elle est sécrétée; elle agit indirectement sur le nombre de cellules de la lignée oligodendrogliale en s'opposant aux effets de l'hyaluronane et en le liant spécifiquement. Ces résultats nous permettent de faire l'hypothèse que l'hyaluronane a un rôle important dans le cerveau en développement sur la lignée oligodendrogliale; que le rapport hyaluronane / hyaluronectine est un facteur important de la myélinisation. L'hypoxie in vitro et la caféine à dose élevée entraînent toutes deux une augmentation de la synthèse cellulaire d'acide hyaluronique, suggérant un effet négatif sur la myélinisation.

O termo neuroplasticidade se refere às mudanças funcionais ou anatômicas que ocorrem no sistema nervoso decorrentes de experiências. O enriquecimento ambiental (EA) é um dos modelos experimentais utilizados para estudar eventos relacionados à neuroplasticidade, pois aumenta a neurogênese, os níveis de neurotrofinas, a sobrevivência neuronal, a sinaptogênese, além de induzir cascatas de sinalização e uma mudança na arborização dendritica dos neurônios de diversas regiões encefálicas. Nos últimos anos a importância dos astrócitos tem ganho destaque, principalmente no que tange a sua capacidade de modular sinapses e na participação ativa em eventos plásticos no encéfalo, essas mudanças estão relacionadas a alterações funcionais e morfológicas. O EA é um modelo interessante para avaliar performances comportamentais pois é um modelo que ocasiona mudanças na capacidade de armazenar e acessar novas informações. Sendo assim, acreditamos que o EA é capaz de gerar efeitos plásticos nos astrócitos do Stratum Radiatum da região CA1 e alterar as respostas comportamentais a tarefa de reconhecimento de objetos. Após oito semanas de EA iniciado logo após o desmame, camundongos CF-1 albinos não apresentam diferenças significativas no número de astrócitos GFAP-ir e na densidade óptica para GFAP no Stratum Radiatum. Entretanto ocorreu um aumento no número de processos originários do soma, aumento no tamanho destes processos no eixo lateral, paralelo as colaterais de Schaffer e no número de intersecções aos círculos concêntricos de Scholl na mesma região. Os astrócitos adquirem um formato estrelado, este achado pode estar relacionado ao aumento da densidade sináptica nesta região e corroboram com a idéia de que modificações astrocitárias são parte ativa dos processos plásticos que ocorrem no encéfalo. Nossos resultados mostraram também uma mudança comportamental na resposta a tarefa de reconhecimento de objetos. Os animais expostos ao EA despendem menos tempo explorando os objetos, tanto familiar como não familiar e apresentam igual capacidade de discriminar os objetos. Estes resultados demonstram um comportamento mais propicio a sobrevivência da espécie em animais expostos ao EA, o que inclui uma rápida exploração e um possível aumento na capacidade de aprender sobre o ambiente.
Environmental enrichment (EE) induces plastic changes in the brain, including morphological changes in hippocampal neurons, with increases in synaptic and spine densities. In recent years, the evidence for a role of astrocytes in regulating synaptic transmission and plasticity has increased, and it is likely that morphological and functional changes in astrocytes play an important role in brain plasticity. In others hand EE is used to investigate behavioral modifications associated with gene-environmental interaction. Our study was designed to evaluate changes in astrocytes induced by EE in the hippocampus, focusing on astrocytic density and on morphological changes in astrocytic processes and the performance in object recognition task (ORT) for evaluate animals ability to learn about their environment. After 8 weeks of EE starting at weaning, CF-1 mice presented no significant changes in astrocyte number or in the density of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in the Stratum Radiatum. However, in the same region occur significant increase in the ramification of astrocytic processes, as well as by an increase in the number and length of primary processes extending in a parallel orientation to CA1 nerve fibers. This led astrocytes to acquire a more stellate morphology, a fact which could be related to the increase in hippocampal synaptic density observed in previous studies. These findings corroborate the idea that structural changes in astrocytic networks are an integral part of plasticity processes occurring in the brain. In other hand, our results indicate that EE decreased the time the animals spent exploring familiar and unfamiliar objects and total time spent exploring both objects, without affecting the capacity of discrimination of objects. These findings indicate a more propitious behavior for species survival in animals subjected to EE, including rapid exploration and learning about the environment.

Dans le systeme nerveux central des vertebres superieurs adultes, la regeneration axonale est extremement limitee. Cependant, il est connu que les neurones de l'hypothalamus mediobasal sont capables de regenerer. Ce travail a donc ete entrepris afin de comprendre les raisons d'une telle reponse postlesionnelle des neurones hypothalamiques. Les travaux realises sur l'hypothalamus ont permis de mettre en evidence par des approches morphologiques des associations preferentielles entre les axones qui regenerent et les prolongements d'un type particulier de cellules gliales present dans l'hypothalamus mediobasal : les tanycytes. Par la suite, l'obtention de cultures enrichies en tanycytes a permis de confirmer leur influence directe sur ces mecanismes de regeneration a travers des interactions adhesives et d'etendre cette capacite vis-a-vis d'une large variete de neurones centraux. Dans la seconde partie de ce travail nous avons donc tente d'identifier certains facteurs susceptibles d'intervenir dans l'effet trophique des tanycytes. Nous avons ainsi montre que, comme les astrocytes embryonnaires (mais contrairement aux astrocytes matures), les tanycytes expriment des quantites importantes de proteines connues pour leur effet neurotrophique, telles l'app (-like), la laminine, la fibronectine, les hspgs et la ncam. Dans un dernier temps, nous avons transplante des tanycytes dans la moelle epiniere lesee, dans le but de favoriser la regeneration des axons supraspinaux. Dans quelques rares cas, la survie des tanycytes transplantes pourrait avoir permis la regeneration d'un certain nombre d'axones afferents leses. L'ensemble de ces resultats indiquent que contrairement aux astrocytes, les tanycytes constituent un support glial qui favorise la regeneration de nombreux types de neurones centraux. Ceci nous permet d'esperer une utilisation des tanycytes a des fins therapeutiques afin de favoriser la regeneration des neurones adultes leses.

The aim of this study was to investigate whether exercise could improve motor behavioral deficits and alter expression of glial fibrillary acidic protein (GFAP) in dorsal striatum in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease (PD). To this end, animals were randomly divided into 4 groups: sham sedentary (SS, n = 7); sham trained (ST, n=8); lesioned sedentary (LS, n=8) and lesioned trained (LT, n = 8). Rats were unilaterally lesioned with 6-OHDA (10 μg/3 μg) injected into the left medial forebrain bundle and sham groups were only injected with vehicle solution. The treadmill training protocol consisted of running with progressive increase in velocity, 5 days/week, during 4 weeks. Behavioral tasks were applied to asses the motor abilities of all animals prior to 6-OHDA injection and at 8th and 29th days post-injection. The tyrosine hydroxylase (TH - in substantia nigra pars compacta) and GFAP (in dorsal striatum) immunostaining was evaluated by semiquantitative analysis of the intensity (optical density - OD). The 6-OHDA lesion decreased the OD of TH and increased the OD of GFAP. In addition, the 6-OHDA lesion increased the number of ipsilateral rotations induced by methylphenidate (40 mg/kg, i.p., 30 min) and caused motor behavioral deficits. On the other hand, the treadmill training resulted in an increase in maximal exercise capacity in both trained groups (ST and LT). The training was able to reduce the number of ipsilateral rotations and ameliorated the motor behavioral deficits on 8th and 29th days postlesion. Interestingly, the exercise led to a significant increase in OD of GFAP in the LT group while there was no such effect in ST group. Our results indicate that treadmill training can improve motor behavioral deficits and suggest that the effects of exercise may be directly or, indirectly, mediated by astrocytes, as an increase in GFAP was observed in the dorsal striatum. Nevertheless, these are the first data showing an increase in GFAP expression post-exercise in this model and further research is needed to determine the precise action of exercise on astrocytes in Parkinson’s disease.

Toute lesion du snc entraine une reaction des neurones et des cellules non neuronales. La regeneration des fibres lesees depend alors autant de leur environnement cellulaire, et plus particulierement de la reaction des cellules gliales, que des facteurs intrinseques. Les astrocytes repondent par une hypertrophie, une hyperplasie et une hyper-expression de proteines de fi (gfap et vimentine) dont le resultat est un enchevetrement cellulaire dense appele cicatrice gliale qui constitue une barriere physique a toute repousse axonale. Le modele de section medullaire complete au niveau t8-t9 nous a permis de mettre en evidence la chronologie de la cicatrice astrocytaire tant au niveau des arnm que de la proteine gfap. La precocite de cette reaction a ete mise en evidence, ainsi que plus tardivement, la dissociation entre arnm et proteine. La reactivite des oligodendrocytes a ete etudiee au moyen de deux marqueurs, cnp et mbp, correspondant respectivement a des stades precoce et tardif de maturation de ces cellules. Nous montrons une difference significative entre la reaction des oligodendrocytes situes au dessus de la section, ou une remyelinisation peut avoir lieu, et ceux situes au dessous. Dans un deuxieme temps, nous avons essaye de limiter la formation de la cicatrice par un traitement utilisant des oxysterols qui inhibent la reactivite des astrocytes. Nous avons realise une hemisection de la moelle chez le rat adulte, nous avons observe que le traitement pharmacologique reduisait l'expression des messagers et de la proteine gfap. La consequence est la mise en evidence d'une repousse axonale (fibres 5ht). Enfin, nous avons voulu savoir si, chez des souris mutantes n'exprimant pas la vimentine, les astrocytes mutes de la moelle etaient toujours aussi reactifs apres lesion. Meme si l'expression de la gfap chez les souris temoin et lesee s'est averee moindre par rapport a la souris sauvage, l'absence de vimentine n'a pas empeche la formation de la cicatrice gliale.

Ratos Wistar machos adultos jovens (6 meses) e velhos (24-25 meses) foram alocados em treinamentos de endurance (corrida em esteira) ou resistido (escalada em grade) durante 6 semanas e comparados a controles sedentários (n=6). Ao final, músculos dos membros posteriores (sóleo e gastrocnêmio) foram analisados por histogramas e atividade total da enzima acetilcolinesterase (AChE). Na medula espinhal lombar, motoneurônios (MN) foram contados, bem como astrócitos da substância cinzenta marcados com GFAP. Densidades ópticas foram medidas nos cornos ventral (CV) e dorsal (CD) para CGRP-ir (MN e CD), AChE e 5-HT-ir (CV e CD). Para histogramas, dados foram analisados usando-se MANOVA e post hoc de Tukey. Os demais dados foram analisados usando-se ANOVA de 1 via e post hoc de Duncan. Resultados: histogramas de ratos jovens mostraram perfis de distribuição distintos após corrida (predominância de fibras de médio diâmetro) ou escalada (predominância de fibras de grande diâmetro) no músculo gastrocnêmio, mas não no sóleo. O perfil de predominância de fibras de pequeno diâmetro observado em ratos velhos sedentários foi igualmente revertido por corrida ou escalada, mas ratos velhos apresentaram limitado aumento de fibras de maior diâmetro. Tanto corrida quanto escalada diminuíram a atividade da AChE muscular. A medula espinhal de animais velhos apresentou menor número de MN e aumento do número de astrócitos. Tanto corrida quanto escalada reduziram a astrogliose no CV, mas não no CD. Ratos velhos mostraram aumento da CGRP-ir em MN, mas nenhum treinamento alterou a CGRP-ir em MN ou no CD. Tanto corrida quanto escalada aumentaram a AChE no CV em todos os grupos treinados. A escalada diminuiu AChE no CD e 5-HT-ir no CV. Nos ratos jovens, a corrida elevou 5-HT-ir no CD, mas não nos ratos velhos. Estes resultados sugerem que distintas modalidades de exercícios crônicos evocam diferentes respostas de neurotransmissores na medula espinhal em diferentes idades.
Young (6 months) and aged (24-25 months) male Wistar rats were assigned to endurance training (ET - treadmill running) and resistance training (RT - grid climbing with increasing weights) during 6 weeks and compared to sedentary controls (n=6). At the end, hindlimb muscles (soleus and gastrocnemius) were analysed by histograms and total AChE activity. In the lumbar spinal cord, motoneurons (MN) were counted, as well as gray matter's GFAP-labeled astrocytes. Optical densities were measured in the ventral (VH) and dorsal (DH) horns for CGRP-ir (MN and DH), AChE staining and 5-HT-ir (VH and DH). Data from histograms were analysed using MANOVA and Tukey’s post hoc. The remaining data Weir analysed using ANOVA and Duncan´s post hoc. Results: gastrocnemius, but not soleus, muscle histograms in young rats showed distinct fiber distribution profiles under ET (toward medium-diameter fibers) and RT (toward large-diameter fibers). The predominance in small-diameter muscle fibers in aged rats was similarly reversed by ET and RT, but aged rats presented limited increase in large-diameter muscle fibers. Both ET and RT decreased muscle AChE activity. The aged spinal cords presented MN loss and greater astrocyte numbers. Both ET and RT reduced astrogliosis in VH, but not in DH. The aged rats displayed elevated CGRP-ir in MN, and neither ET nor RT altered CGRP-ir in MN or DH. Exercise (ET and RT) markedly increased AChE staining in VH in all groups. RT decreased AChE in DH and 5-HT-ir in VH. In young rats, running elevated 5-HT-ir in the DH. These results suggest that different chronic exercise modalities and age evoke distinct spinal cord neurotransmitter responses.

INTRODUÇÃO: A restrição do crescimento intrauterino (RCIU) é conhecida como uma condição na qual o feto não consegue desenvolver todo o seu potencial de crescimento intrínseco. Crianças com RCIU em idade escolar têm prejuízos no aprendizado, memória, atenção e cognição em geral, e maior predisposição a transtornos psicológicos como Transtorno de Déficit de Atenção e Hiperatividade (TDAH), quando comparados com crianças não restritas. OBJETIVO: este estudo busca encontrar modificações neurobiológicas encefálicas, em ratos Wistar adolescentes, que possam explicar os efeitos, em parâmetros comportamentais, de neuroplasticidade e memória, da RCIU por redução da pressão de perfusão uterina (RPPU). MÉTODOS: ratas Wistar prenhes de 75 dias de idade foram submetidas à RPPU no dia gestacional 14, período que corresponde ao inicio da fase de rápido crescimento, diferenciação e maturação em ratos, por meio da colocação de um clipe na artéria aorta abdominal descendente antes da bifurcação para as artérias ilíacas e um clipe em cada artéria ovariana. O grupo Sham foi submetido à laparotomia exploratória sem a RPPU e o grupo controle não foi submetido a nenhum procedimento cirúrgico. Ratos Wistar de 30 dias, provenientes de mães submetidas à RPPU, Sham ou Controles, foram submetidos ao Teste de Campo Aberto, para avaliação da atividade locomotora e exploratória, e no 32º dia pós-natal (DPN) foram submetidos ao teste de Reconhecimento do Novo Objeto para avaliar a memória de longo prazo. Os encéfalos da prole foram coletados no 34º DPN e conservados para análise. A Técnica de imunoistoquímica foi realizada para mensurar a expressão de marcadores celulares neurais: NeuN (neurônios maduros) GFAP (astrócito) no córtex pré-frontal e estriado da prole. RESULTADOS: a RPPU causou diminuição no peso ao nascimento no grupo RCIU e Sham, porém não houve diferença no peso entre os grupos no 30º DPN. A RPPU aumentou significativamente a duração da gestação no grupo RCIU em um dia. Os animais restritos apresentaram aumento na atividade exploratória no teste de campo aberto. RCIU e Sham apresentaram déficit de memória no teste de reconhecimento do novo objeto. Associado a essas alterações comportamentais, a RCIU levou a o aumento significativo na expressão de NeuN no CPF e de GFAP no estriado no 34º DPN. CONCLUSÃO: a RPPU produz alterações na gravidez causando maior duração da ges-tação e RCIU na prole, conduzindo a modificações comportamentais e de memória, assim como a nível encefálico celular, promovendo novas perspectivas sobre as consequências do modelo em ratos de RCIU-RPPU. São necessários mais estudos para desvendar se essas modificações continuam presente na vida adulta dos ratos.
INTRODUCTION: Intrauterine growth restriction (IUGR) is known as a condition where the fetus cannot develop its intrinsic growth potential. IUGR Children in early scholar age have impaired learning abilities, memory, attention and cognitive deficit in general; also, they are in higher risk of psychological disorders as Attention Deficit and Hyperactivity Disorder (ADHD) when compared with non-IUGR children. OBJECTIVE: The study aims to find neurobiologi-cal brain modifications, in adolescent Wistar rats, that could clarify these memory and behav-ioral impairments in a rat animal model of IUGR by Reduced Uterine Perfusion Pressure (RUPP). METHODS: 75 days-old pregnant Wistar rats were underwent to RUPP procedure on 14th gestational day, period where the fast growing, differentiation and maturation phase begins in rats, by putting one clip at the abdominal descendent aorta before iliac bifurcation and one clip in each ovarian artery. The Sham group underwent exploratory laparotomy procedure without the RUPP and the control group was not exposed to any surgical procedure. 30 days old Wistar rats from RUPP, Sham, and Control mothers were exposed to the Open Field Test to assess the locomotor and exploratory activity and, at 32th postnatal day (PND), to the Novel Object Recognition test to assess the long-term memory. Offspring brains were collected at 34th PND and preserved for analysis. Immunohistochemistry technique was performed to measure the expression of neural cells markers: NeuN (mature neurons) and GFAP (astrocytes) in prefrontal cortex and striatum on the offspring. RESULTS: RUPP caused a decreased birth weight in IUGR and Sham group, however there was no difference between birth weight at 30th PND. RUPP significantly increased gestation in 1 day in IUGR rats. IUGR animals had greater exploratory, locomotor activity in the Open Field test. IUGR and Sham showed memory deficit in the Novel Object Recognition test. Associated to those behavioral modifications, the RUPP leaded to a significant increase in expression of NeuN and GFAP marker in prefrontal cortex and striatum at PND34. CONCLUSIONS: The RUPP dis-turbs pregnancy increasing gestation length and inducing IUGR in the offspring; which leads to behavioral and memory modifications, also, in a brain cellular level, promoting new insight about the IUGR-RUPP rat model consequences. Further analysis are needed to find whether this changes are still present in adult life.

Les séquelles neurologiques secondaires aux atteintes de la substance blanche du prématuré constituent un problème de santé publique. La physiopathologie de ces atteintes fait appel aux facteurs associés à la prématurité. Parmi ces facteurs, l'implication du retard de croissance intra-utérin est hautement débattue. Les effets du RCIU sur le développement de la substance blanche ont été étudiés dans un modèle chez le rat. Les ratons ayant un RCIU sévère développent à la naissance des lésions diffuses de la substance blanche similaires à celles des prématurés humains. Ces atteintes associent réaction gliale et déficit persistant de la myélinisation. Le RCIU modéré est associé à une atteinte transitoire de la substance blanche. Le retour à la normal de la myélinisation dès la deuxième semaine de vie est concomitant avec une augmentation de la prolifération oligodendrocytaire. De plus, le préconditionnement hypoxique associé au RCIU modéré protège la substance blanche vis-à-vis d'une injection intra-corticale d'un agent glutamatergique excitotoxique. D'autre part les conséquences d'un RCIU sévère sur le cortex ont également été étudiées. Chez les rats adultes ayant subi un RCIU sévère, une perte de densité neuronale corticale est détectée, en particulier un déficit de neurones GABAergiques. Cette perte est associée à des troubles comportementaux. Ainsi ces modèles miment la plupart des atteintes histologiques et comportementales des enfants prématurés et de petit poids de naissance. L'ensemble de ces travaux contribue à mieux comprendre le rôle des facteurs (hypoxie, ischémie, déplétion en facteurs trophiques) associés au RCIU dans la genèse des lésions du cerveau immature
Behavioural deficit secondary to white matter damage of preterm infant is a major public health issue. The risk-factors associated with prematurity play a role in the pathophysiology of these insults. Among these factors, the implication of intra-uterine growth restriction (IUGR) is still debated. The effect of IUGR on white matter development were studied in a model of prenatal growth restriction in rat. Similarly to preterm infants, severe growth restricted pups exhibit diffuse white matter damage associating glial reaction and protracted myelination defect. Moderate IUGR is associated with transcient white matter insult in rat pups. The myelination repair is concomitant with the enhancement of oligodendrocytes proliferation after the first week of life. Furthermore, hypoxic preconditionning associated with moderate IUGR is protective against a postnatal excitotoxic insult in a "double-hit" protocol. In another hand, the impact of IUGR on cortical development were investigated. In severely growth restricted adult rat, a decrease of neuronal density, and particularly of GABAergic sub-populations density, was detected in the somato-sensory cortex. This neuronal deficit was associated with behavioral and cognitive disorders. Thus, these animal models of IUGR in rat mimick the histologic and behavioural deficit of preterm infants with low birth-weight. This study provides new elements for a better comprehension of the role of factors (hypoxia, ischemia, trophic factors deprivation) associated with IUGR in the pathophysiology of immature brain damage

Oxaliplatin is a platinum-organic drug with anti-neoplastic properties used for colon-rectal cancer. In respect to other platinum derivates, oxaliplatin induced only a mild hematological and gastrointestinal damage. Its limiting side effects are the neurotoxicity and the subsequent nerve hyperexcitability whom result in a neuropathic syndrome. In a rat model of oxaliplatin-induced neuropathy we underlined a significant oxidative stress induced by the chemotherapic agent. Silibinin and α-tocopherol, two well known natural antioxidants, prevented the oxidative damage and, at same time, reduced neuropathic pain [1]. Aimed to deep inside the mechanism of oxaliplatin neurotoxicity we developed a cellular model using rat astrocytes, glia cells significantly activated in neuropathic rats repeatedly treated with oxaliplatin [2]. In cell cultures, oxaliplatin-evoked redox unbalance led to the activation of the apoptotic process evaluated by caspase-3 activity, a central effector of the controlled cell death. 10 µM silibinin and α-tocopherol protected astrocytes from oxaliplatin-induced caspase-3 activation. To evaluate the safety of these compounds towards the oxaliplatin anti-tumoral efficacy, apoptosis signal was analyzed in HT-29, a human colorectal cancer cell line. Interestingly, the antioxidant compounds were completely inactive against oxaliplatin-dependent apoptosis in HT-29. These evidences suggested different mechanisms by which oxaliplatin induces apoptosis in tumoral rather normal cells. In this view our data suggested that oxaliplatin treatment induced the intrinsic apoptosis activation in astrocytes normal cells. In tumoral cells, the extrinsic apoptotic pathway was prevailing. In this perspective, new agents candidate to treat oxaliplatin neuropathy, should possess protective properties against mithocondrial disfunctions. On the other hand, the chemotherapy anticancer efficacy could be maintained preserving the extrinsic apoptotic pathway activation. References: 1) Di Cesare Mannelli et al. Oxaliplatin-induced neuropathy: Oxidative stress as pathological mechanism. Protective effect of silibinin. The Journal of Pain, Vol 13, No 3 (March), 2012 Mar;13(3):276-84 2) Di Cesare Mannelli et al. Oxaliplatin-induced oxidative stress in nervous-derived cellular models. Could it correlate with in vivo neuropathy?. Free Radic Biol Med. 2013 Mar 30 31; 61C:143-150

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
Os astrócitos, a maior população celular do cérebro, têm um papel importante no processamento e metabolismo neuronal e no controlo da barreira hematoencefálica. Evidências apoiam a existência de uma comunicação bidirecional entre neurónios e astrócitos no controlo da função cerebral, originando o conceito de "sinapse tripartida", que postula que os astrócitos são o terceiro elemento ativo das sinapses, modulando a plasticidade sináptica. A plasticidade sináptica, nomeadamente a potenciação de longa duração (LTP, “long-term potentiation”) e a depressão de longa duração (LTD, “long-term depression”) representam os mecanismos neurofisiológicos associados à memória, na qual o hipocampo tem um papel crucial. A doença de Alzheimer (DA) caracteriza-se por uma acumulação de placas amilóides extracelulares, compostas maioritariamente por péptidos β-amilóide (Aβ) e por tranças neurofibrilares constituídas por proteína tau hiperfosforilada. Assim, Aβ é considerado causar DA, levando às perdas e disfunção sináptica subjacentes aos défices cognitivos associados a esta patologia neurodegenerativa. No entanto, o papel dos astrócitos, nomeadamente na atividade sináptica na DA continua pouco esclarecido. O presente estudo pretende definir o impacto dos astrócitos na plasticidade sináptica hipocampal, particularmente em LTP e LTD, bem como avaliar marcadores astrocíticos, em condições não-patológicas e de DA. Para mimetizar a DA, as fatias de hipocampo foram incubadas com o peptídeo Aβ1-42 solúvel (exposição aguda, 50 nM, durante 40 min) ou administrado intracerebroventricularmente (icv, 0,5 mM) em murganhos C57Bl/6 jovens adultos. Além disso, também foi usado o modelo animal triplo transgénico da DA, os murganhos 3xTgAD.Realizaram-se registos de eletrofisiologia nos neurónios piramidais presentes na região CA1 na via proveniente dos colaterais de Schaffer e mediu-se LTD primeiramente em condições de exposição aguda a Aβ1-42. Os resultados obtidos demonstram que a Aβ1–42 teve um impacto robusto na amplitude de LTD, levando a um desvio de LTD para LTP. Para silenciar a contribuição dos astrócitos na modulação da plasticidade sináptica, utilizámos a gliotoxina L-α-aminoadipato (L-AA), previamente validada na indução da patologia astrocítica. A incubação de fatias do hipocampo com L-AA (100 µM, 2 h) não teve efeito na amplitude LTD em condições não-patológicas (controlo). No entanto, a exposição a L-AA em condições patológicas de DA reverteu significativamente os efeitos de Aβ1-42 na plasticidade sináptica, levando a uma recuperação da amplitude de LTD.Os animais injetados (icv) foram sujeitos a testes comportamentais para avaliar o desempenho de tarefas dependentes do hipocampo e, os murganhos injetados (icv) com Aβ1-42 apresentaram défices significativos de memória quando comparados com os injetados com veículo. À semelhança dos efeitos observados em condições de exposição aguda a Aβ1-42, a LTD e também LTP foram significativamente comprometidas em comparação com os murganhos injetados com veículo. Adicionalmente fez-se estudos imunohistoquímicos para identificar as proteínas astrocíticas, proteína acídica fibrilar glial (GFAP) e S100β, em cortes transversais de fatias do hipocampo de murganhos injetados (icv). Os resultados revelaram um aumento na imunorreatividade da GFAP em animais Aβ1-42. Em concordância com os estudos de exposição aguda a Aβ1-42, a incubação com L-AA apenas teve efeito na amplitude de LTD sob condições que mimetizam a DA (Aβ1-42 icv), revertendo significativamente os efeitos de Aβ1-42 na plasticidade sináptica, recuperando o prejuízo na LTD. Contrariamente, em condições não-patológicas o L-AA diminuiu significativamente a LTP, sendo este efeito menos evidente em condições de DA (Aβ1-42 icv). Ademais, a gliotoxina (L-AA) aumentou a imunorreatividade de GFAP observado em condições controlo (veículo icv) e não tendo efeito significativo na reatividade dos astrócitos em condições de DA. Estes dados reforçam a interferência desta gliotoxina na função astrocítica e a ideia de que os astrócitos são cruciais para a regulação da plasticidade sináptica, em especial na LTD em condições de DA.A plasticidade sináptica foi avaliada em murganhos 3xTgAD (11 meses) onde se observou uma tendência para um aumento na amplitude LTP comparado com murganhos não transgénicos (nonTg). Além disso, as fatias de hipocampo de murganhos 3xTgAD exibiram uma intensa reatividade astrocítica avaliada pela imunorreatividade de GFAP nas regiões CA1 e CA3. A disfunção astrocítica induzida por L-AA pareceu reverter as alterações em LTP observadas em 3xTgAD. Em fatias de ambos os grupos de animais a incubação com L-AA causou um aumento da GFAP.Este trabalho apresenta fortes evidências de que os astrócitos estão disfuncionais em condições de DA, comprometendo a plasticidade sináptica hipocampal e a memória, suportando o conceito de que os astrócitos podem ser um apropriado alvo para o desenvolvimento de novos tratamentos para a DA.
Astrocytes, the largest cell population in the brain, have a key role in neuronal function and metabolism and in the control of blood brain barrier. Increasing evidences support the existence of a bidirectional communication between neurons and astrocytes in the control of brain function, giving rise to the concept of ‘tripartite synapse’, which postulates that astrocytes are the third active element of synapses with the capacity of fine-tune synaptic plasticity. The synaptic plasticity, such as the long-term potentiation (LTP) and long-term depression (LTD), constitute the neurophysiological mechanisms of memory, a process in which hippocampus has a key role. Alzheimer’s disease (AD) is characterized by the accumulation of extracellular amyloid plaques, composed mainly by amyloid-β (Aβ) peptide, and by neurofibrillary tangles constituted by hyperphosphorylated tau protein. Aβ peptides are considered to be a causative agent of AD, which can cause synaptic loss and dysfunction that underlie the cognitive and memory deficits associated to this neurodegenerative disorder. However, the role of astrocytes, namely on synaptic function under AD conditions, is still not completely understood. The present study aims to define the impact of astrocytes on hippocampal synaptic plasticity, in particular on LTP and LTD, as well as to evaluate the astrocytic markers in physiological and AD-like conditions. To mimic AD-like conditions, hippocampal slices were incubated with soluble Aβ1–42 (acute exposure, 50 nM, for 40 min) or administrated intracerebroventricularly (icv, 0.5 mM) in young adult C57Bl/6 mice. Moreover, a transgenic AD animal model, 3xTgAD mice, was used.Electrophysiological recordings in the Schaffer collaterals-CA1 pyramid synapses were performed and LTD was first measured under acute exposition to Aβ1–42. The data obtained showed that Aβ1–42 had a robust impact on LTD amplitude, leading to a shift of LTD toward LTP, compared with control mice. To silence astrocytic contribution in shaping synaptic plasticity we used the gliotoxin L-α-aminoadipate (L-AA), previously validated to induce astrocyte pathology. Incubation of hippocampal slices with L-AA (100 µM, 2 h) had no effect on LTD amplitude in non-pathological conditions (control). However, treatment with L-AA under AD-like conditions significantly reverted the effects of Aβ1–42 on synaptic plasticity, rescuing LTD impairment.The icv-injected mice were behaviorally characterized using hippocampal-dependent tasks, and it was observed a memory deficit in icv Aβ1-42 mice when compared with vehicle mice. Similarly, to that observed in hippocampal slices acutely exposed to Aβ1–42, in icv Aβ1-42 injected mice the hippocampal LTD and also LTP were significantly compromised, as compared with icv-vehicle mice. Furthermore, immunohistochemical analysis probing for astrocytic markers, such as the glial fibrillary acidic protein (GFAP) and S100β were performed in transverse hippocampal sections obtained from slices of icv-vehicle and Aβ1–42 injected mice. The results showed an increase in GFAP immunoreactivity in slices from icv Aβ1–42 injected mice. In agreement with our data obtained in conditions of acute Aβ1–42 exposure, the incubation of hippocampal slices with the gliotoxin (L-AA) had no effect on LTD in control conditions. However, under AD-like conditions (icv injection), L-AA significantly reverted the effects of Aβ1–42 on synaptic plasticity, rescuing LTD impairment. By contrast in non-pathological conditions, the LTP was significantly decreased by L-AA, being this effect less evident in icv Aβ1–42 injected mice. Moreover, it was observed that this gliotoxin (acute exposure) increased hippocampal GFAP immunoreactivity in physiological conditions, but in AD-like conditions decrease the effect was the opposite. These data reinforce that indeed this gliotoxin was interfering with astrocytes function and these glial cells had a prominent role in shaping synaptic function, contributing to synaptic plasticity impairment, mainly LTD, in AD-like conditions.We also evaluated the hippocampal synaptic plasticity in the 3xTgAD mice (with 11 months old) that showed a tendency to an increase in hippocampal LTP amplitude compared with littermates non-transgenic (nonTg) mice. Moreover, the slices from 3xTgAD exhibited an increased astrocytic reactivity, assessed by GFAP immunoreactivity in CA1 and CA3 regions of hippocampus. The astrocytes blunting triggered by L-AA tend to rescue the LTP alterations observed in 3xTgAD. The incubation of LAA of hippocampal slices from both 3xTgAD and nonTg mice seemed to cause an increase in GFAP immunoreactivity.Overall, the current work demonstrates strong evidences that in AD-like conditions, the astrocytes are dysfunctional, impairing hippocampal synaptic plasticity and memory. Thus, these studies support that astrocytes can be viewed as a valid target for the development of novel treatments for AD.
Outro - 016684-PTDC/NEU-NMC/4154/2014 (POCI-01-0145-FEDER-016684) - Papel dos astrócitos no controlo da memória - foco nos recetores de adenosina A2A - Fundação para a Ciência e Tecnologia
Outro - COMPETE POCI-01-0145-FEDER-007440 - Trabalho multidisciplicar no âmbito da neurociência cognitiva na saúde e na doença - Fundação para a Ciência e a Tecnologia.

Dissertação de mestrado em Ciências da Saúde
Schizophrenia is a debilitating psychiatric disorder that affects approximately 1% of the population and is characterized by psychotic events, as well as cognitive and negative symptoms, namely impairments in social interaction. The discovery of drugs that are able to reduce the psychotic symptoms of the disorder helped to shed some light on the mechanisms involved in the etiology of schizophrenia. However, the high complexity of the disease along with the adverse side effects associated with antipsychotic drugs seem to build a long road to the desired treatment of this severe disorder. Functional and structural studies have reported abnormalities in multiple brain areas, namely the hippocampus, one of the particular regions in which the proliferation of stem cells occurs in the adult brain. Importantly, impairments in adult neurogenesis have been described in the brains of schizophrenic patients. However, the possible role of newly formed glial cells and astrocytic plasticity in the etiology of this disorder and in the effects of antipsychotic drugs remains largely unknown. In the present study, a neurodevelopmental animal model of schizophrenia based in the prenatal exposure to alkylating agent methylazoxymethanol (MAM) was used to evaluate the impact of different classes of antipsychotic (AP) drugs in gliogenesis, astrocytic remodeling and social behavior in rats. Animals exposed to MAM in the prenatal period (GD17) were treated in adulthood with a first generation AP (haloperidol), two second generation AP´s (clozapine and risperidone) and a third generation AP (aripiprazole). The results revealed significant impairments in social behavior, gliogenesis and astrocytic morphology in animals prenatally exposed to MAM. While chronic treatment with aripiprazole was able to revert the impairments in social behavior and restore the levels of gliogenesis, the first and second generation AP´s haloperidol, clozapine and risperidone revealed a specific effect in restoring the detrimental effects of MAM exposure in astrocytic morphology and complexity. However, no significant differences in the expression of astrocytic-related genes were observed in the hippocampus. In conclusion, these results suggest that gliogenesis and astrocytic plasticity may play a key role in social behavior in the context of schizophrenia. Furthermore, the effects of different classes of AP drugs in these phenomena may pave a new way in the treatment of the negative symptoms of schizophrenia.
A esquizofrenia é uma doença psiquiátrica debilitante, que afeta aproximadamente 1% da população e que é caracterizada por fenómenos psicóticos, bem como por sintomas cognitivos e negativos, nomeadamente distúrbios em interação social. A descoberta de fármacos que reduzem os sintomas psicóticos da doença contribuiu para o avanço no conhecimento de mecanismos afetados no contexto da esquizofrenia. No entanto, a alta complexidade da doença aliada aos efeitos secundários adversos dos antipsicóticos afastam a possibilidade de um tratamento eficaz para esta grave doença. Estudos funcionais e estruturais mostram que várias áreas estão afetadas nesta doença, nomeadamente o hipocampo, uma das poucas estruturas onde a proliferação de células estaminais ocorre no cérebro adulto. Curiosamente, já foi mostrado que a neurogénese adulta encontra-se afetada nos cérebros de pacientes com esquizofrenia. No entanto, o papel de células gliais recentemente formadas e plasticidade de astrócitos na origem desta doença bem como os efeitos de antipsicóticos a este nível é ainda altamente desconhecido. Neste estudo, o modelo animal de esquizofrenia de injeção pré-natal do agente alquilante acetato de metilazoximetanol (MAM), foi usado para avaliar o impacto de antipsicóticos (AP) de diferentes classes em gliogénese, remodelação de astrócitos maduros e comportamento social em rato. Animais expostos a MAM durante o período pré-natal (GD17) foram tratados em idade adulta com um AP de primeira geração (haloperidol), dois de AP’s de segunda geração (clozapina e risperidona) e um AP de terceira geração (aripiprazole). Os resultados mostram distúrbios significativos ao nível do comportamento social, da gliogénese e da morfologia astrocítica em animais expostos a MAM durante o período pré-natal. Embora o tratamento crónico com aripiprazole tenha permitido reverter os distúrbios em comportamento social e recuperar os níveis gliogénese, os AP’s de primeira e segunda geração haloperidol, clozapina e risperidona mostraram um efeito específico relativamente à recuperação dos efeitos prejudiciais da exposição a MAM na morfologia e complexidade astrocítica. No entanto, não foram encontradas diferenças significativas relativamente à expressão de genes relacionados com astrócitos no hipocampo. Em conclusão, estes resultados indicam que a gliogénese e plasticidade astrocítica parecem estar a ter um papel fundamental ao nível de comportamento social no contexto da esquizofrenia. Além disso, os efeitos de fármacos AP de diferentes classes nestes fenómenos podem ajudar a construir um novo caminho para o tratamento dos sintomas negativos da esquizofrenia.

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 muM-1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.

In this research project we studied signal transduction pathways triggered by cholinergic system and involved in short term and long term memory formation in the CA1 region of the rat hippocampus, focusing on mTOR pathway. We also evaluated memory impairments and molecular and cellular alterations of the neuron-astrocyte-microglia triad in the hippocampus, in three different animal models of neurodegeneration: normal brain aging, acute neuroinflammation induced by LPS infusion and chronic cerebral hypoperfusion induced by bilateral common carotid artery occlusion

Les grands prématurés sont particulièrement vulnérables aux lésions inflammatoires de la substance blanche (WMI) qui augmentent le risque de troubles cognitifs et neurodéveloppementaux à long terme dans cette population. L’utilisation de l’imagerie par résonance magnétique (IRM) dans cette population a permis une évaluation non invasive de la progression des WMI et une meilleure compréhension de la pathologie. Les WMI sont associées une activation de la microglie et des astrocytes et la production de facteurs pro-inflammatoires, dont l’interleukine 1 (IL-1). En utilisant un modèle de WMI induite par injection intracérébrale de lipopolysaccharides (LPS), nous avons évalué dans un premier temps les changements de méthylation de l’ADN durant la phase aigüe (24 h) et la phase chronique (21 jours) de l’inflammation. Par la suite, nous avons déterminé la capacité de l’IRM multimodale de détecter la lésion et la réponse thérapeutique à un antagoniste du récepteur de l’IL-1. Finalement, par le biais d’un antagoniste et d’un modulateur allostérique du récepteur à l’IL-1, nous avons évalué in vitro le rôle de la signalisation IL-1 durant la phase aigüe de la modulation de l’activation de la microglie et des astrocytes par le LPS. Nous avons démontré la présence d’une altération du méthylome cérébral dans divers mécanismes liés au neurodéveloppement et à la réponse immunitaire. De plus, l’application de l’IRM multimodale dans notre modèle a permis d’évaluer in vivo la lésion et le début de la réponse thérapeutique durant la phase aigüe (24 h) de l’inflammation. L’évaluation à l’IRM corrèle aux changements observés par immunomarquage post mortem. In vitro, le LPS induit une réponse mixte de la microglie et des astrocytes qui évoluent dans le temps vers une réponse pro-inflammatoire et neurotoxique. Bien que l’IL-1 est hautement exprimée par la microglie et les astrocytes, son inhibition a un effet limité sur la modulation de l’activation gliale dû à la multitude de voies activées par le LPS durant la phase aigüe de l’inflammation.
Very premature infants are particularly vulnerable to inflammatory white matter injury (WMI) which increases the risk of long-term cognitive and neurodevelopmental disorders in this population. The use of magnetic resonance imaging (MRI) in this population has allowed non-invasive assessment of the progression of WMI and a better understanding of the pathology. WMI is associated with activation of microglia and astrocytes and the production of pro-inflammatory mediators, including interleukin 1 (IL-1). Using a model of inflammatory WMI induced by intracerebral injection of lipopolysaccharides (LPS), we first evaluated the changes in DNA methylation during the acute phase (24 h) and the chronic phase (21 days) of inflammation. We then determined the ability of multimodal MRI to detect the lesion and the therapeutic response to an IL-1 receptor antagonist. Finally, using an antagonist and an allosteric modulator of the IL-1 receptor, we evaluated in vitro the contribution of IL-1 signaling during the acute phase of the modulation of microglia and astrocytes activation by LPS. We have shown the presence of persistent alteration DNA methylation profile in the brain that was associated with pathways involved in neurodevelopment and immune response. In addition, the application of multimodal MRI in our model made it possible to evaluate in vivo the lesion and the therapeutic response during the acute phase (24 h) of the inflammation. The changes at the MRI correlated to post-mortem evaluation by immunostaining. In vitro, LPS induce a mixed response of microglia and astrocytes which evolved over time toward a pro-inflammatory and neurotoxic phenotype. Although IL-1 is highly expressed by microglia and astrocytes, its inhibition has a limited effect on the modulation of glial activation due to the multitude of pathways activated by LPS during the acute phase of inflammation.

No
Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 microM) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

Tese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Methamphetamine (METH) is a powerful psychostimulant drug of abuse that has gained worldwide popularity, and its use originates severe health problems. Despite extensive characterization of METH-induced neurotoxicity over the last years, many questions remain unanswered. Several reports have demonstrated that oxidative stress, mitochondrial dysfunction, and neuroinflammation are some of the neurotoxic features of METH. More recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Additionally, it is well known that astrocytes play a crucial role in modulating BBB structure and function, as well as in regulating brain water content. However, the effect of METH on the crosstalk between brain endothelial cells (ECs) and astrocytes has never been addressed before. Also, water fluxes that take place between the different compartments of the brain, and between brain parenchyma and the blood are highly controlled. Thus, disturbances in this well-regulated homeostasis cause brain edema, which will have deleterious effects on brain function. Importantly, the water transport at BBB is regulated by water channels, aquaporins (AQPs), and AQP4 is the most important at the Central Nervous System, being express on astrocytic endfeet in contact with brain vessels. Brain edema is a hallmark of several neuropathologies, and METH consumption is not an exception. Yet, to date, nothing is known about the role of AQP4 under METH conditions. Furthermore, AQP4 has two isoforms, M1 and M23, and the ratio M1/M23 regulates water homeostasis since M23 stabilizes the channel function but M1 disrupts the AQP4 structure. Taking into consideration all the gaps in this field, it is urgent to clarify the role of AQP4 in METH-induced BBB dysfunction and brain edema formation. The present thesis is divided into 5 chapters. In chapter 1 is presented a review of the literature about the different themes that were explored in the laboratory and detailed in the following chapters. In chapter 2, the impact of METH on astrocytes-ECs crosstalk was investigated with a particular interest in the role of tumor necrosis factor alpha (TNF-α). After observing that METH increased TNF-α released by both astrocytes and ECs, it was also proved that this proinflammatory cytokine was responsible for endothelial permeability through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. These in vitro results were corroborated by animal studies showing an increase of BBB permeability and TNF-α levels in the mice striatum, which was prevented by NF-κB pathway blockade. Overall, it was shown that TNF-α/NF-κB signaling pathway has a key role in METH-induced BBB dysfunction. Next, in chapter 3, it was investigated the direct effect of METH on AQP4 system concluding that METH, besides BBB dysfunction, is also able to induce a cytotoxic brain edema and depressive-like behavior. Curiously, AQP4 was shown to have a predominant role of such alterations since its inhibition prevented all the effects observed in mice. Moreover, AQP4 via reactive oxygen species (ROS) production was involved in cell swelling and altered astrocyte morphology triggered by METH since AQP4 knockdown or its pharmacological blockade, as well as an antioxidant treatment (namely vitamin C) were able to prevent METH effects in astrocytes. In conclusion, AQP4 was identified as a new target against METH-induced neurogliovascular dysfunction and depressive-like behavior. Following the results observed in chapter 2 and 3, a new strategy to counteract the negative effects of METH was applied by using a natural flower product. Thus, in chapter 4, it was proved that parthenolide (PTL), a feverfew plant extract, has an anti-inflammatory role and prevented METH-induced BBB permeability and brain edema. Additionally, TNF-α via activation of its receptor 1 (TNFR1) was involved in astrocytic swelling induced by METH. In sum, PTL plays a beneficial role against neuroinflammation and neurogliovascular dysfunction triggered by METH. Finally, in chapter 5, a general discussion is presented. Overall, the present work shows that METH interferes with brain water homeostasis and BBB function, culminating in behavioral abnormalities. Moreover, both neuroinflammation and oxidative stress are involved in such negative effects of METH, and new strategies to counteract these deleterious consequences were identified, such as AQP4 blockade and the use of PTL.
A metanfetamina (MET) é uma droga de abuso muito viciante com grande popularidade mundial, e que causa sérios problemas de saúde. Apesar da extensa caracterização da sua neurotoxicidade nos últimos anos, muitas questões continuam sem resposta. Alguns estudos têm mostrado que o stresse oxidativo, a disfunção mitocondrial e a neuroinflamação são alguns dos efeitos nefastos da MET. Mais recentemente demonstrou-se que a MET interfere com a função normal da barreira hematoencefálica (BHE), causando alterações na homeostase da água o que pode levar a uma situação de edema cerebral. Para além disso, sabe-se também que os astrócitos têm um papel muito importante na modulação da estrutura e função da BHE, bem como na regulação do conteúdo de água cerebral. No entanto, o efeito da MET na comunicação entre as células endoteliais (CEs) e os astrócitos nunca foi estudado anteriormente. Por outro lado, o movimento de moléculas de água entre os diferentes compartimentos do cérebro e entre o parênquima cerebral e a corrente sanguínea ocorre de forma controlada. Assim, distúrbios nesta homeostase irão causar uma situação de edema, o qual terá um impacto negativo na função cerebral. O transporte de água na BHE é regulado por canais de água, denominados aquaporinas (AQPs), sendo que a AQP4 é a mais importante no Sistema Nervoso Central, e encontra-se expressa nas terminações dos astrócitos que contactam com os vasos cerebrais. De facto, o edema cerebral ocorre em muitas neuropatologias, e o consumo de MET não é exceção. No entanto, o papel da AQP4 nos efeitos da MET é ainda desconhecido. Além disso, a AQP4 tem duas isoformas, a M1 e a M23, e é a sua proporção que regula a homeostase da água, uma vez que a presença da isoforma M23 estabiliza a função do canal de água enquanto a isoforma M1 causa alterações na função da AQP4. Deste modo, é importante esclarecer o papel da AQP 4 na disfunção da barreira hematoencefálica e na formação do edema cerebral induzidos por MET. A presente tese está dividida em 5 capítulos. No capítulo 1 é apresentada uma revisão da literatura sobre os diversos temas estudados no laboratório e detalhados nos capítulos seguintes. No capítulo 2 investigou-se o efeito da MET na comunicação entre astrócitos e CEs com particular interesse no papel do fator de necrose tumoral alfa (TNF-α). Depois de mostrar um aumento da libertação de TNF-α induzido por MET, quer pelos astrócitos quer pelas CEs, provou-se que esta citocina pró-inflamatória estava envolvida no aumento da permeabilidade das CEs através da ativação da via de sinalização do fator nuclear kappa B (NF-κB). Estes resultados foram corroborados por estudos em animais onde se observou um aumento da permeabilidade da BHE e dos níveis de TNF-α no estriado de murganho, efeitos estes que foram prevenidos pelo bloqueio da via do NF-κB. Deste modo, conclui-se que a via de sinalização do TNF-α/NF-κB está envolvida na disfunção da BHE induzida por MET. De seguida, no capítulo 3 avaliou-se o impacto direto da MET no sistema da AQP4 e foi possível demonstrar que esta droga de abuso, para além de induzir uma disfunção da BHE, também originou um edema cerebral citotóxico e comportamento do tipo depressivo. Curiosamente, a AQP4 teve um papel predominante nestas alterações já que o seu bloqueio preveniu todos os efeitos observados nos murganhos. In vitro foi também possível comprovar o papel importante da AQP4 via produção de espécies reactivas de oxigénio já que o silenciamento deste canal de água ou a sua inibição farmacológica, bem como a exposição a um antioxidante (vitamina C) preveniram as alterações morfológicas induzidas pela MET nos astrócitos. Em conclusão, a AQP4 foi identificada como um alvo importante para prevenir as alterações neurogliovasculares e comportamento depressivo induzidos por MET. Na sequência dos efeitos negativos da MET observados nos capítulos 2 e 3, colocou-se a hipótese de uma nova abordagem com um produto natural de origem vegetal. Deste modo, no capítulo 4 concluíu-se que o partenolídeo (PTL), um extrato obtido da artemísia dos prados (Tanacetum parthenium), tem um papel anti-inflamatório e preveniu o aumento da permeabilidade da BHE e formação de edema cerebral induzidos por MET. Mais ainda, foi possível demonstrar que o TNF-α, através da ativação do seu recetor TNFR1, estava envolvido no aumento de volume dos astrócitos observado na presença de MET. Assim, este trabalho permitiu concluir que o PTL tem um feito benéfico em condições de neuroinflamação e disfunção neurogliovascular induzidos por MET. Por último, o capítulo 5 inclui uma discussão geral sobre os resultados obtidos nos capítulos anteriores. Em conclusão, esta tese permitiu mostrar que a MET interfere não só com a homeostase da água no cérebro, mas também com a função da BHE, e que estes efeitos podem conduzir a alterações comportamentais. Para além disso, demonstrou-se ainda que a neuroinflamação e o stresse oxidativo estão subjacentes aos efeitos negativos causados pela MET e foram identificadas duas abordagens para prevenir estes efeitos, tais como o bloqueio da AQP4 e o uso do partenolídeo.

Research Doctorate - Doctor of Philosophy (PhD)
Schizophrenia is a debilitating disorder of neurodevelopmental origins that likely stems from the cumulative action of a range of genetic and environmental factors. Epidemiological evidence has identified maternal infection during gestation as one significant environmental risk factor for the development of the disorder. Evidence from animal models has further validated the link between maternal immune activation (MIA) in the absence of an active infection and the later life development of schizophrenia-like pathology in the offspring. In particular, work in mouse models has suggested that the gestational time at which MIA occurs can alter the behavioural and neurobiological phenotype displayed. Specifically, that MIA in late gestation is involved in schizophrenia-relevant cognitive dysfunction and altered NMDA receptor expression, whereas MIA in early gestation is more closely associated with behavioural deficits reminiscent of positive symptomology and dopaminergic neurotransmission. The aim of the current thesis was to extend the mouse findings to another species, the rat, and further explore the effects of MIA. In addition to producing a reliable rat model of schizophrenia where distinct behavioural and neurological phenotypes associated with schizophrenia are produced following MIA at either early or late gestational time-points (gestational day 10 or 19, respectively), the current thesis extends on previous work by examining the schizophrenia biomarker of mismatch negativity and assessing the neuroinflammatory state of offspring. Behavioural assessments revealed that MIA in either early or late gestation produced transient impairments in working memory and reductions in PPI. In these behavioural studies, there was no clear distinction between a dopamine and glutamate-related behavioural phenotype based on the gestational timing of exposure. However, early but not late gestation MIA did produce alterations in the dopaminergic system of males, as indicated by increased dopamine 1 receptor mRNA in the nucleus accumbens. EEG experiments demonstrated that although the male rat brain is able to generate human-like (adaptation-independent) mismatch responses (MMRs), and although MIA (regardless of gestational timing) does alter MMRs, it does not do so in a manner comparable with schizophrenia. Immunohistochemical techniques revealed that MIA does result in subtle neuro-immune changes in adult offspring, with an increase in microglial immunoreactivity identified in the frontal white matter of late, but not early, gestation MIA animals. Furthermore, a strong trend towards increased astrocyte immunoreactivity that approached significance was identified in the prefrontal cortex of late, but not early MIA offspring. The combined results have demonstrated that MIA during the chosen gestational time-points are sufficient to disrupt neurodevelopmental processes producing long-term alterations in behavioural and neuropathological measures relevant to schizophrenia. However, the phenotype characterised here deviates slightly from previous findings from mouse models indicating potential differences in the critical periods of neurodevelopmental susceptibility to MIA exposure between the rat and mouse. Importantly this research has provided insights into the underlying neuro-immune changes which may contribute to the behavioural abnormalities seen in adult MIA offspring and has provided evidence that MIA in rats can alter the prominent schizophrenia relevant electrophysiological biomarker of adaptation-independent MMRs, providing a basis to further investigate these measures and their underlying mechanisms.