Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento. / Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group. We also showed an increase in the number of NADPH-d stained cells in the dentate gyrus in the HI group, at all ages, as also an increase in the intensity of staining in the Ammons horn (CA1 and CA3) at P9 in the HI group and, after that, a decrease in this staining at P23 and P90 in this same group. In the behavioral tests we observed a decrease in the motor activity in the HI group with a partial recovery all over the several sessions in the ROTAROD test, however this group did not reach the same performance as the SHAM group. HI animals did not show a higher level of anxiety when compared to SHAM animals, ruling out the hypothesis that anxiogenic factors may be impairing the results in the motor behavior tests. Our results showed that the model of uterine arteries clamping could be an important tool in the study of the effects of perinatal HI, by producing several consequences that are very similar to the effects observed in newborn children who suffered an HI event.

O núcleo motor do trigêmeo (Mo5) está cercado por um anel de neurônios pré-motores localizados na região h. Estudos demonstram que neurônios que inervam o Mo5 estão distribuídos no tronco encefálico e no prosencéfalo. Após implante de traçador retrógrado no Mo5, verificamos células retrogradamente marcadas no núcleo mesencefálico do trigêmeo (Me5), na região h e em núcleos prosencefálicos como o central da amígdala (CeA), a área hipotalâmica lateral (LH) e o parasubtalâmico (PSTh). Para confirmação, realizamos injeção de traçador anterógrado e investigamos, também, a neuroquímica das projeções. Neurônios do CeA que se projetam para o Mo5 recebem inervação de fibras imunorreativas ao fator liberador de corticotrofina (CFR-ir) e/ou à tirosina hidroxilase (TH-ir); alguns neurônios da LH que se projetam para o Mo5 são imunorreativos à orexina (ORX) e alguns neurônios do PSTh que se projetam para o Mo5 são innervados por fibras TH-ir. O Me5 recebe grande inervação do CeA e moderada da LH e do PSTh, possuindo grande aferência de fibras imunorreativas ao CRF, ORX e TH / The trigeminal motor nucleus (Mo5) is surrounded by a ring of premotor neurons defined as the h region. Studies have shown that neurons innervating the Mo5 are located in brainstem and in forebrain nuclei. Through the injection of the retrograde tracer cholera toxin b subunit/CTb in the Mo5, we found retrograde labeled neurons in the brainstem including the h region and the mesencephalic trigeminal nucleus (Me5), and in forebrain nuclei such as the central nucleus of amygdala (CeA), the lateral hypothalamic area (LH) and the parasubthalamic nucleus (PSTh). As control, we injected the anterograde tracer biotin dextran amine and found that these areas project direct or indirectly via the h region or the Me5 to the Mo5. Some CeA neurons that project to the Mo5 receive corticotrophin releasing factor (CRF) and tyrosine hydroxylase (TH) innervation, some LH neurons that project to Mo5 express orexin, and PSTh neurons that project to the Mo5 receive TH innervation. The Me5 is also innervated by CeA, LH and PSTh neurons and by CRF, orexin and TH immunoreactive fibers

The purpose of this thesis project was to examine the effects of chronic alcohol on the excitability and molecular adaptation of GABA neurons of the ventral tegmental area (VTA). GABA neurons are of interest with regards to ethanol intoxication, reinforcement, and dependence due to their widespread distribution and connectivity to mesocorticolimbic dopamine (DA) neurons implicated in alcohol reward and addiction. Since we have previously shown adaptation of VTA GABA neuron firing rate to chronic ethanol (Gallegos, Criado et al. 1999) and suppression of gap-junction (GJ) mediated coupling between these neurons by acute ethanol (Stobbs, Ohran et al. 2004), we wanted to further characterize the effects of chronic ethanol on VTA GABA neuron excitability, electrical coupling and molecular adaptation. In particular, we analyzed the GJ mediated coupling and protein regulation of VTA GABA neurons following a three week period of continuous ethanol exposure via liquid diet. Although some animals showed tolerance, there was no significant tolerance to ethanol inhibition of GJ-mediated electrical coupling. In addition, we were able to characterize differences in mRNA expression levels for the DA synthesizing enzyme tyrosine hydroxylase (TH), the DA D2 receptor and the NMDAR2B receptor subunit in DA versus GABA neurons, all three of which were expressed at higher levels in DA neurons. We also determined the effects of chronic ethanol on mRNA levels of these same proteins as well as μ-opioid receptors (μORs) and connexin-36 (Cx36) GJs. Most significantly, we found a down-regulation of the DA D2 receptor, confirming that molecular modification occurs in these VTA GABA neurons with chronic alcohol. While we reject our hypothesis that acute ethanol inhibition of VTA GABA neuron electrical coupling would undergo tolerance to chronic ethanol in these non-dependent rats, which was the focus of this thesis, it remains to be determined if tolerance to chronic ethanol might be obtained in ethanol-dependent rats.

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<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

El objetivo general de este trabajo ha sido estudiar la posible implicación de diferentes cascadas de proteín kinasas en las modificaciones cardiacas que se producen tras la administración de naloxona a ratas dependientes de morfina. Los datos obtenidos indican que durante la abstinencia a morfina se produce un aumento del turnover de NA, de la actividad TH y de su fosforilación en serina 40 y 31, lo que sugiere la puesta en marcha de mecanismos post-transcripcionales. Por otra parte, la vía de la PKA estaría implicada en el incremento del turnover de NA, en el aumento de TH total y en la fosforilación y activación de TH en serina 40 durante dicho síndrome. Por último, la vía de la PKC sería una de las vías implicadas en la expresión de c-fos, así como la de las ERK, que estaría también implicada en la activación de TH en serina 31. / The main aim of this work was to study the posible involvement of different protein kinases in the cardiac adaptive changes induced during morphine withdrawal. Our results show an increase of NA turnover, TH activity and TH phosphorylation at serine 31 and 40, suggesting starting post-trascriptional mechanisms. On the other hand, PKA transduction system could be implicated in the enhanced NA turnover, in the total TH increase and in the phosphorylation and activation of TH at serine 40 during this syndrome. Finally, PKC pathway would be involved in c-Fos expression as well as ERK system which would also be responsible for TH phosphorylation at serine 31.

Tyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.